Compounds that inhibit human DNA ligases and methods of treating cancer

ABSTRACT

Methods for treating cancer using compounds that inhibit human DNA ligases. Methods for using compounds that inhibit human DNA ligases to provide insights into the reaction mechanisms of human DNA ligases, for example to identify the human DNA ligase involved in different DNA repair pathways. Screening methods for compounds that inhibit human DNA ligases.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 13/864,916 filed Apr. 17, 2013, now U.S. Pat. No. 9,073,896, which is a divisional of U.S. patent application Ser. No. 12/576,410 filed Oct. 9, 2009, now U.S. Pat. No. 8,445,537, which is a continuation-in-part of International Patent Application No. PCT/US2008/059931 filed Apr. 10, 2008, which claims benefit of U.S. Provisional Application 60/911,000, filed Apr. 10, 2007, all applications of which are herein incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with the support of the U.S. government under Grant Numbers GM057479, GM047251, ES012512, CA092584 and CA102428 from National Institutes of Health (NIH). The U.S. government has certain rights in this invention.

NAMES OF PARTIES OF A JOINT RESEARCH AGREEMENT

Not Applicable

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Not Applicable

BACKGROUND OF THE INVENTION

Despite many years of research, there still exists a compelling need to develop novel and more effective therapeutic strategies for human cancer. The use of many agents used in cancer treatment is limited because of their cytotoxic effects on normal tissues and cells. This is a particular concern for agents that kill cells by damaging DNA and/or inhibiting DNA replication. Moreover, it is becoming more and more evident that the simultaneous or sequential attack on different aspects of cancer cell metabolism by combinations of agents is more effective than the use of a single agent. This highlights the need to develop a wider variety of therapeutic agents that hit different molecular targets in cancer cells.

As mentioned above, therapeutic agents such as ionizing radiation and temozolomide, which damage DNA, have cytotoxic effects on normal tissues and cells as well as cancer cells. Despite the frequent use of agents that either damage DNA or inhibit DNA replication, there are relatively few available compounds that specifically target DNA repair and/or DNA replication-related proteins (1-3). Topoisomerase-I inhibitors, for example alter the capacity of a key DNA replication enzyme to proceed along an entire chromosome. Although cytotoxic, this class of compounds is currently being used to treat human cancer. There are also inhibitors of DNA damage response proteins, including the checkpoint kinase Chk1, poly(ADP) ribose polymerase, DNA dependent protein kinase, ATM kinase, MGMT and AP endonuclease (1-3) that are in preclinical or early clinical evaluation as cancer therapeutics. Applicants specifically contemplate that certain types of inhibitors of DNA repair pathways will have therapeutic utility because they will potentiate the cytotoxic effects of other treatments of cancer, for example, ionizing radiation and chemotherapeutic agents that damage DNA. This may permit specific targeting of tumors and/or the use of lower doses of DNA damaging agent, thereby reducing toxicity to normal tissues and cells. In addition, there is evidence that the DNA repair capabilities of cancer cells may be different than those of normal cells. For example, BRCA2-deficient cells established from individuals with an inherited predisposition to breast cancer are extremely sensitive to inhibitors of poly (ADP-ribose) polymerase because they are defective in homologous recombination (4,5). Thus, inhibitors of DNA repair proteins may specifically target cancer cells as compared to normal cell populations.

Under normal circumstances, the genome is propagated and maintained by the combination of a highly accurate DNA replication machinery and a network of DNA repair pathways. The increased incidence of cancer associated with DNA repair-deficient human syndromes illustrates the role of these pathways in protecting against deleterious genetic changes that contribute to cancer formation. There is growing interest in the identification of DNA repair inhibitors that will enhance the cytotoxicity of DNA-damaging agents because combinations of DNA-damaging agents and DNA repair inhibitors have the potential to concomitantly increase the killing of cancer cells and reduce damage to normal tissues and cells if either the damaging agent or the inhibitor could be selectively delivered to the cancer cells (2). Because DNA ligation is required during replication and is the last step of almost all DNA repair pathways, DNA ligase-deficient cell lines exhibit sensitivity to a wide range of DNA-damaging agents (6). Thus, DNA ligase inhibitors may have pleiotropic effects on cell proliferation and sensitivity to DNA damage.

DNA ligases catalyze the joining of interruptions in the phosphodiester backbone of double-stranded DNA, making them essential enzymes for DNA repair and replication. In addition, they are an indispensable reagent in molecular biology research for generating recombinant DNA. DNA ligases are members of the larger nucleotidyl transferase family that also includes RNA ligases and mRNA capping enzymes. In the first step of the ligation reaction, DNA ligases react with a nucleotide co-factor, either NAD+ or ATP, to form the covalent enzyme-AMP intermediate. Next the AMP moiety is transferred to the 5′ phosphate termini in duplex DNA, forming the DNA adenylate intermediate. Finally, the non-adenylated enzyme catalyzes phosphodiester bond formation between the 3′ hydroxyl and 5′ phosphate termini.

BRIEF SUMMARY OF THE INVENTION

Because of their involvement in DNA replication and DNA repair, DNA ligase inhibitors are likely to be anti-proliferative and to potentiate the cytotoxicity of DNA damaging agents, properties that may have clinical utility in the treatment of cancer, in particular malignancies with an altered DNA damage response. DNA joining by a DNA ligase is required to link together Okazaki fragments during DNA replication and to complete almost all DNA repair pathways (6). Applicants' invention involves inhibitors of DNA ligases that: (i) inhibit cell growth and/or kill cells; (ii) potentiate the cytotoxic effects of many DNA damaging agents that introduce a wide variety of different types of DNA lesions, including agents such as ionizing radiation, topoisomerase inhibitors (irinotecan and topotecan), PARP inhibitors, 5-fluorouracil, mitomycin C, bleomycin, melphalan, cyclosphosphamide, platinum compounds, cis-platinum and BCNU (Carmustine) and temozolomide that are currently being used to treat human cancer; and/or (iii) potentiate the cytotoxic effects of pro-apoptotic agents and inhibitors of signaling pathways that promote proliferation, for example a bcr-abl kinase inhibitor (imatinib (GLEEVAC)) that may be used to treat human cancer.

The combination therapy methods of the present invention are contemplated to be administered at the same time or at separate times to a subject in need thereof.

Applicants' invention includes methods for screening of individual chemical compounds, a synthetic chemical collection and/or a natural product library to identify compounds that inhibit human DNA ligase.

The human genome contains three genes, LIG1, LIG3 and LIG4, which encode ATP-dependent DNA ligases (6). These enzymes have unique cellular functions, but they also appear to have some functional redundancy. Although these enzymes have a conserved catalytic domain and use the same reaction mechanism, they are directed to participate in different DNA transactions by specific protein-protein interactions (6). DNA ligase I (hLigI) has a major role in the joining of Okazaki fragments during DNA replication and also in DNA excision repair. Based on the cellular phenotype of human DNA ligase I-deficient cells (7-9), specific inhibitors of DNA ligase I should inhibit cell growth and/or kill cells. Since cancer cells in general tend to be more proliferative than normal cells, it is conceivable that they will be more susceptible to the effects of DNA ligase I inhibitors. In support of this, it has been shown that human cancer lines grown either in vitro or as tumors in nude mice and human tumor specimens have elevated levels of DNA ligase I (10). In addition, human DNA ligase I deficient cells are hypersensitive to DNA alkylating agents and an inhibitor of poly(ADP-ribose) polymerase (PARP), 3 aminobenzamide. (8-10). Applicants' invention involves DNA ligase I specific inhibitors that will preferentially kill rapidly proliferating cancer cells and potentiate the cytotoxic effects of DNA alkylating agents such as temozolomide and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) that are used clinically, and, when used in combination with PARP inhibitors (several of which are in clinical trials), will synergistically enhance cytotoxicity.

Several distinct DNA ligase polypeptides that function in nuclear DNA repair, mitochondrial DNA metabolism and germ cell development are encoded by the LIG3 gene (6). It appears that all cells express a single mRNA, DNA ligase IIIα mRNA that encodes mitochondrial and nuclear forms of DNA ligase IIIα. In addition, there is an alternative splicing event in male germ cells that generates DNA ligase IIIβ. Nuclear DNA ligase IIIα participates in the repair of DNA single strand breaks and the short patch subpathway of base excision repair. This appears to underlie the sensitivity of DNA ligase III-deficient cells to DNA alkylating agents and ionizing radiation (11,12). Applicants' invention also involves DNA ligase III inhibitors that will potentiate the cytotoxic effects of DNA alkylating agents such as temozolomide and BCNU, and ionizing radiation, all of which are used to treat human cancer. Furthermore, it has been shown that depletion of human DNA ligase III by RNAi disrupts mitochondrial function (13,14), raising the possibility that DNA ligase III inhibitors may cause cytotoxic effects via effects on mitochondrial function and may potentiate the effects of pro-apoptotic drugs. Since many tumors cells have alterations in energy metabolism, it is possible that they may be more susceptible to agents that target mitochondria. There is emerging evidence that DNA ligase I and DNA ligase IIIα participate in different subpathways of base and nucleotide excision repair and that these subpathways may be, at least in a part, functionally redundant (15,16). Applicants' invention also involves compounds that inhibit both DNA ligase I and DNA ligase III. These compounds will potentiate the cytotoxicity of DNA damaging agents such as 5-fluorouracil, temozolomide, BCNU, platinum compounds and mitomycin C that introduce lesions that are removed by excision repair to a greater extent than compounds that are specific for either DNA ligase I or DNA ligase III.

The LIG4 gene product, hLigIV, completes the repair of DNA double strand breaks (DSBs) by nonhomologous end joining (NHEJ) and V(D)J recombination events that generate diversity in immunoglobulin and T-cell receptor loci during immune system development (6). Notably, DSBs are the major cytotoxic lesion introduced by ionizing radiation, which is the most commonly used modality in the treatment of human cancer. Furthermore, NHEJ is the major DSB repair pathway in human cells and inactivation of the LIG4 gene has a more severe effect than inactivation of genes encoding proteins that act earlier in the NHEJ pathway (17), suggesting that, once DSBs are committed to the NHEJ pathway, they cannot be recovered and repaired by pathways utilizing either DNA ligase I or DNA ligase III. Applicants' invention also involves DNA ligase IV inhibitors that will dramatically potentiate cell killing by ionizing radiation and radiomimetic drugs.

In the complex formed by hLigI on DNA with a non-ligatable nick, three hLigI domains encircle and interact with the nicked DNA duplex. (18) Two of these domains, an adenylation domain (AdD) and an OB-fold domain (OBD), are present in other DNA ligases and nucleotidyl transferases. In contrast, the DNA binding domain (DBD, residues Asp262 to Ser535) is restricted to eukaryotic ATP-dependent DNA ligases (6). Notably, the DBD is the predominant DNA binding activity within hLigI and stimulates joining in trans by a hLigI fragment containing the adenylation and OB-fold domains (18). Based on these properties, Applicants chose to focus on identifying compounds that bind to the DBD and inhibite hLig1 activity by interfering with DNA binding.

Applicants' invention also includes the following.

Methods for using the inhibitors to delineate the molecular mechanisms of the DNA ligation reaction.

Methods for identifying the DNA ligase that completes different DNA repair pathways in cell extracts.

Methods for identifying the pathways involved in the repair of different DNA lesions using the ligase inhibitors in cell extract and cell culture assays.

Methods for treating cancer using compounds that inhibit human DNA ligases by inhibiting cell growth, killing tumorous cells and/or potentiate cell killing by DNA damaging agents.

Methods for treating cancer using compounds that inhibit human DNA ligase including treating epithelial-derived cancers, such as epithelia neoplams selected from the colon cancer, lung cancer, breast cancer, GI cancer, ovarian cancer and head and neck cancer.

Methods for treating cancer using compounds that inhibit human DNA ligase including treating hematological malignancies, such as hematological malignancy selected from the chronic and acute leukemias.

Methods for treating cancer using compounds that inhibit human DNA ligase including treating hematological malignancies, such as lymphomas.

The methods for treating cancer using compounds that inhibit human DNA ligase including treating skin cancer, melanoma, gliablastoma, neuroblastoma, sarcoma, colon cancer, breast cancer, cervical cancer, prostate cancer, pancreatic cancer, ovarian cancer, esophageal cancer, stomach cancer and lung cancer.

Identification and Characterization of Human DNA Ligase Inhibitors

One research focus has been to determine the cellular functions of the DNA ligases encoded by the human LIG1, LIG3 and LIG4 genes and to define the molecular mechanisms by which these enzymes are directed to participate in specific DNA transactions. Recently the structure of the catalytic domain of human DNA ligase I complexed with nicked DNA was determined (18). This was the first structure of a eukaryotic DNA ligase and the first structure of any DNA ligase complexed with DNA.

Attempts to identify human DNA ligase inhibitors by screening of chemical and natural product libraries have met with limited success. Previous attempts to identify DNA ligase inhibitors have involved in vitro screening of a chemical and a natural product library (19,20). Several compounds that inhibit DNA ligase I have been identified, but these compounds have not been extensively characterized in terms of their specificity and mechanism of action. A problem with screening of random chemical libraries using an in vitro DNA ligation assay is that it yields two classes of non-specific inhibitors; (i) molecules that bind to the DNA substrate; (ii) nucleotide analogs that inhibit not only ATP-dependent DNA ligases but also other nucleotide-dependent cellular enzymes. To circumvent these problems, Applicants used available structural information identifying the interfaces between DNA ligase I and nicked DNA (18) to design an in silico screening method for the identification of small molecules that disrupt the physical and functional interactions between DNA ligase and nicked DNA. In developing the screening methods, Applicants identified a binding pocket between residues His337, Arg449, and Gly453 that are located in the central region of the DBD and make direct contacts with the DNA substrate. Other residues comprising the binding site include Gly448, Arg451 and Ala455. (18, 21, 34-37) Based on amino acid sequence conservation, Applicants determined that it is likely that this binding pocket is relatively well conserved in human DNA ligases III and IV.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the Structures of 9 of Applicants' inhibitors of human DNA ligases.

FIGS. 2A and 2B show the Specificity of Compounds 67, 82 and 189 as human DNA ligase inhibitors. FIG. 2A shows the IC50 values determined for Compounds 67, 82 and 189 using the fluorescence-based DNA joining assay for each of DNA ligases I, III and IV and T4 DNA ligase. FIG. 2B shows the effect of Compounds 67, 82 and 189 on DNA joining by human DNA ligases I, III and IV and T4 DNA ligase as determined using the radioactive gel-based assay.

FIGS. 3A, 3B and 3C show the mechanism of action of human DNA ligase inhibitors. FIG. 3A shows Human DNA ligases I, III and IV and T4 DNA ligase incubated with [α³²P] ATP in the absence or presence of Compounds 67, 82 and 189 (100 μM). After separation by SDS-PAGE, the labeled ligase-AMP complex was detected by phosphorimaging. FIG. 3B shows labeled ligase-adenylate form of human DNA ligases I, III and IV and T4 DNA ligase incubated with a linear DNA substrate containing a single non-ligatable nick in the absence or presence of Compounds 67, 82 and 189 (100 μM). After separation by denaturing gel electrophoresis, the labeled DNA-AMP complex was detected by phosphorimaging. FIG. 3C shows non-adenylated human DNA ligases I, III and IV and T4 DNA ligase incubated with labeled DNA-AMP in the absence or presence of Compounds 67, 82 and 189 (100 μM). After separation by denaturing gel electrophoresis, labeled ligated DNA was detected by phosphorimaging.

FIGS. 4A, 4B and 4C show the effect of human DNA ligase inhibitors on replication and repair by human cell extracts. FIG. 4A shows a cell extract from the human cervical cancer cell line HeLa incubated with the indicated labeled flap substrate that mimics a common intermediate in DNA replication and long path base excision repair in the absence or presence of Compounds 67, 82, 184 and 189 (25 μM). After separation by denaturing gel electrophoresis, labeled fragments corresponding to the DNA substrate, cleaved product and fully repaired product were detected by phosphorimaging (28). FIG. 4B shows a HeLa cell incubated with labeled dTTP and the indicated linear substrate with an incised AP site that mimics an intermediate in short path base excision repair in the absence or presence of Compounds 67, 82 and 189 (25 μM). Lane 6, DNA ligase I was immunodepleted from the extract prior to the assay. Lane 7, DNA ligase III was immunodepleted from the extract prior to the assay. After separation by denaturing gel electrophoresis, labeled fragments corresponding to a single nucleotide insertion and fully repaired product were detected by phosphorimaging. FIG. 4C shows a HeLa extract incubated with a linear cohesive-ended 1 kb fragment with cohesive ends repair in the absence or presence of Compounds 67, 82 and 189 (25 μM). Lane 7, DNA ligase I was immunodepleted from the extract prior to the assay. Lane 8, DNA ligase III was immunodepleted from the extract prior to the assay. Lane 8, DNA ligases III and IV were immunodepleted from the extract prior to the assay. After separation by native agarose gel electrophoresis, the linear DNA substrate, re-circularized substrate and dimers trimers etc of the 1 kb substrate were detected by staining with ethidium bromide.

FIGS. 5A and 5B show the effect of DNA ligase inhibitors on cell growth in the absence or presence of DNA damage. FIG. 5A shows different concentrations of Compounds 67 and 151 added to asynchronous subconfluent populations of HCT116 and MCF10A cells. FIG. 5B shows asynchronous subconfluent populations of HCT116 and MCF10A cells pre-treated with different concentrations of Compound 64 prior to the addition of 3 aminobenzamide (2 mM), MMS (100 μM) or cis-platinum (1 μM). After incubation for 5 days, cell growth was measured by the MTT assay (16).

FIGS. 6A, 6B, 6C and 6D show the cytotoxicity of DNA ligase inhibitors in the absence or presence of DNA damage. FIG. 6A shows different concentrations of Compound 184 were added to asynchronous subconfluent populations of HCT116, MCF7 and MCF10A cells. Colonies were counted after two weeks. FIG. 6B shows different concentrations of Compound 151 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells in the absence or presence of MMS (50 μM). Colonies were counted after two weeks. FIG. 6C shows different concentrations of Compound 189 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells. After 1 hour, cultures were irradiated with 2 gray of ionizing radiation. Colonies were counted after two weeks. FIG. 6D shows different concentrations of Compound 67 were added to asynchronous subconfluent populations of HCT116, MCF7 and MCF10A cells. After 1 hour, cultures were irradiated with 2 gray of ionizing radiation. Different concentrations of Compound 67 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells in the absence or presence of MMS (50 μM). Colonies were counted after two weeks.

FIG. 7. Michaelis-Menten analysis of ligase inhibitors. Effect of ligase inhibitors on DNA-protein complex formation by hLigI. A, hLig1 (0.05 pmol) was incubated in the absence (o) and presence of L189 (left), L67 (middle), and L82 (right) at 25 μmol/L (open triangle) and 50 μmol/L (open square) with increasing amounts of a linear nicked DNA substrate. Lineweaver-Burk double reciprocal plots of initial reaction velocity (1/V) versus substrate concentration (1/S). B, a labeled linear substrate with a single nonligatable nick (1 pmol) was incubated with no addition (lane 1), 0.25 pmol of hLigI (lanes 2 and 3), 0.5 pmol of hLigI (lanes 4 and 5), and 1 pmol of hLigI (lanes 6 and 7) in the absence (−) or presence (+) of 100 μmol/L of L189. C, a labeled linear substrate with a single nonligatable nick (1 pmol) and hLigI (3 pmol) were incubated with either no addition (lane 2) or 100 μmol/L (lane 3), 60 μmol/L (lane 4), 50 μmol/L (lane 5), 30 μmol/L (lane 6), 20 μmol/L (lane 7), or 10 μmol/L (lane 8) of L82. Lane 1, 1 pmol of DNA substrate alone. The positions of the labeled DNA substrate and DNA-protein complexes are indicated.

FIG. 8. Characterization of the cytostatic effect of L82. A, MCF10A (▪), MCF7 (♦), HCT116 (▴), and HeLa (upsidedown triangle) cells were plated in the absence or presence of L82 (left), L67 (middle), and L189 (right). After 6 days, cell viability was measured and is expressed as a percentage of the value obtained with untreated cells. B, MCF7 cells were plated out in the absence or presence of L82 (top) and L67 (bottom) at the indicated concentrations. After 2 weeks, colonies were stained with crystal violet. C, after serum starvation for 4 days, MCF 7 cells were returned to serum-containing media either without (▪) or with 50 μmol/L of L82 (●). The cell cycle distribution at various time intervals was determined by fluorescence-activated cell sorting. D, asynchronous populations of MCF cells were either untreated (top) or treated with L82 at 10 μmol/L (middle) and 50 μmol/L (bottom). After 3 days, tubulin and DNA were visualized by fluorescence microscopy (bars, 0.5 mm).

FIG. 9. L67 and L189 are cytotoxic and potentiate the cytotoxic effects of DNA-damaging agents: altered levels of DNA ligase in cancer cells. A, effect of L67 (left) and L189 (right) on the survival of MCF7 (Δ), HCT116 (□), HeLa (⋄), and MCF10A (upsidedown triangle) cells. B, normal breast epithelial MCF10A cells (open symbols) and breast cancer MCF7 cells (filled symbols) in the absence (circles) or presence of 3 μmol/L of L67 (squares) were exposed to increasing concentrations of MMS (left). Normal breast epithelial MCF10A cells (open symbols) and colon cancer HCT116 cells (filled symbols) in the absence (circles) or presence of 20 μmol/L of L189 (squares) were exposed to increasing doses of ionizing radiation (right). C, hLigI (I), hLigIIIα (III), and hLigIV (IV) were detected in extracts (400 μg) of the indicated cell lines by immunoblotting. To control for extract loading β-actin was also detected by immunoblotting.

FIG. 10. The DNA substrate (thin tube) is encircled by three domains of human DNA Ligase I, i.e. the DNA binding domain (DBD) containing residues Asp262-Ser535 (connected large tubes-bottom), the adenylation domain (AdD) Pro536-Asp748 (wide ribbon-upper right), and the OB-fold domain (OBD) Tyr749-Ser901 (narrow ribbon-upper left). The AMP cofactor (in CPK representation) is located in AdD and the putative binding site on DBD is represented by red spheres and the three residues defining the binding pocket, His337, Arg449 and Gly453, are shown in CPK representation.

FIG. 11. Distributions of (a) the molecular weight and (b) the van der Waals attractive energy Va of the 50,000 compounds selected, via different normalizations by N the number of heavy atoms of the compound, from the preliminary screening. The normalization Va/N^(2/3), as shown by the blue dotted line is used to select compounds.

FIG. 12. (A) Alignment of the crystal structure and four conformations selected from the 20 ns MD simulation. (B) Orientations of three of the residues lining the binding site.

FIG. 13, Distributions of (a) the molecular weight and (b) the total interaction energy, Etot, of the 1000 compounds selected via different normalization schemes from the secondary docking.

FIG. 14. Distributions of physical and molecular properties of the 233 selected compounds.

FIG. 15. Assessment of inhibitor and DNA interaction by EtBr replacement assay. There is no binding of 82, 67 and 189 with DNA (A). In contrast, EtBr is replaced to DNA binding by 192 in a concentration (0, 50, 100 μM) dependant manner (B).

FIG. 16. The distribution of the N^(2/5) normalized total energy scores for the top 1000 compounds (black circles), the selected 233 compounds (red squares) and the 10 compounds that specifically inhibit hLigI activity by >50% (spikes). The scores for the 3 characterized compounds are shown as longer spikes; the wider spikes are associated with two compounds having similar scores.

DETAILED DESCRIPTION OF THE INVENTION Materials and Methods

Chemicals.

Compounds identified by CADD screening were purchased from ChemBridge, ChemDiv, MayBridge, MDD, Nanosyn, Specs, Timtec, and Tripos. 189 was from Specs, and 82 and 67 were from Chemdiv. Tenmillimolar stocks were prepared in DMSO and stored at −20° C. The molecular masses of three compounds 67, 82 and 189 were confirmed by mass spectrometry in the University of Maryland School of Pharmacy facility.

Proteins.

Recombinant human DNA ligase I was purified as described previously (22, 29). T4 DNA ligase was purchased from NEB.

DNA Joining Assays.

Candidate ligase inhibitors identified by CADD were assayed for their ability to inhibit hLigI and T4 DNA ligase using a high-throughput, fluorescence energy transfer-based DNA joining assay (22). Duplicate reactions (30 μL) containing 10 pmol of nicked DNA substrate and either 0.25 pmol of hLigI or 10 units of T4 DNA ligase were incubated in the presence or absence of 100 μmol/L of the putative inhibitor. DNA binding by the candidate DNA ligase inhibitors was measured by displacement of ethidium bromide from DNA as previously described (30).

A radioactive gel-based DNA ligation assay was performed as previously described (22). A 25-mer (5′-CGC CAG GGT TTT CCC AGT CAC GAC C-3′), and a 5′-[³²P] end-labeled 18-mer (5′-GTA AAA CGA CGG CCA GTG-3′) were annealed to a complementary 44-mer oligonucleotide, generating a linear duplex with a central nick. DNA joining reactions (30 μL) containing 0.5 pmol of labeled DNA substrate, and hLigI (0.02 pmol), hLigIIIβ (0.02 pmol), hLigIV/XRCC4 (0.1 pmol), or T4 DNA ligase (0.02 pmol) in ligation buffer were incubated in the absence or presence of ligase inhibitors at 25° C. for 30 min.

Assays for steps 2 and 3 of the ligation reaction.

To analyze step 2 of the ligation reaction, labeled ligase-AMP intermediates (10 pmol (22)) were incubated overnight at 25° C. with an unlabeled nonligatable version (dideoxy residue at the 3′-terminus of the nick) of the DNA oligonucleotide substrate (10 pmol), either in the presence or absence of the ligase inhibitors (100 μmol/L).

To analyze step 3 of the ligation reaction, an adenylated labeled version of the 18-mer was prepared as described (24). The DNA substrate containing a preadenylated nick (0.5 pmol) and hLigI (0.05 pmol), hLigIIIβ (0.05 pmol), hLigIV/XRCC4 (0.1 pmol), or T4 DNA ligase (0.05 pmol) were incubated in ligation buffer without ATP, either in the presence or absence of the ligase inhibitors (100 μmol/L). Reactions were stopped by the addition of an equal volume of gel loading dye (95% formamide, 0.05% bromophenol blue, and 0.05% xylene cyanol). After heating at 95° C. for 5 min, DNA was separated by denaturing polyacrylamide gel electrophoresis. Labeled oligonucleotides were detected and quantitated in the dried gel by phosphorImager analysis (Molecular Dynamics).

Kinetic Analysis of Ligase Inhibitors.

To measure the initial rates of ligation, hLigI (0.05 pmol) was incubated with 0.5 to 100 pmol of the fluorescent, nicked DNA substrate and various concentrations of the ligase inhibitors. K_(i) values were obtained from Lineweaver-Burk double reciprocal plots and curve-fitting using PRISM v3.03 (GraphPad).

Electrophoretic Mobility Shift Assay.

A labeled linear duplex with a nonligatable nick was incubated with hLig1 in ligation buffer (30 μL total volume) with or without ligase inhibitors for 120 min at 25° C. After the addition of an equal volume of native gel buffer [160 mmol/L Tris-HCl (pH 6.8), 20% glycerol, 1.4 mol/L 2-mercaptoethanol, and 0.05% bromophenol blue], samples were separated by electrophoresis through a 12% native polyacrylamide gel and detected in the dried gel by phosphorImager analysis.

Cell Extract Assay of DNA Replication and Repair.

Extracts were prepared from human cervical cancer HeLa cells as described previously (27, 31). For base excision repair (BER) assays, the extraction buffer contained 100 mmol/L of KCl, whereas for nonhomologous end-joining (NHEJ) assays, extraction buffer contained 400 mmol/L of KCl. Where indicated, DNA ligases were immunodepleted from the extracts as described (32) using protein A or G Sepharose beads (GE Healthcare) and anti-Lig1, anti-LigIII (GeneTex), or anti-LigIV (ABCAM) antibodies. Depletion was confirmed by immunoblotting.

A labeled 5′-flap substrate (0.1 pmol; (31)) was incubated with 20 μg of extract in the absence or presence of ligase inhibitors (100 μmol/L) at 25° C. for 5 min in ligation buffer (final volume, 50 μL). For short patch BER, a linear duplex containing a single uracil residue was preincised by treatment with uracil DNA glycosylase and APE1 (both from NEB) to generate a strand break with 3′ hydroxyl and 5′ deoxyribose phosphate termini. Reactions (50 μL) containing 0.3 pmol of the incised DNA substrate, 10 μCi of [α³²P]dTTP, and 20 μg of extract either in the absence or presence of ligase inhibitors (100 μmol/L) were incubated at 25° C. for 2 min in ligation buffer. After separation by denaturing polyacrylamide gel electrophoresis, labeled oligonucleotides were detected in the dried gel by phosphorImager analysis (Molecular Dynamics).

To assay NHEJ (24), a 1 kb end-labeled BamHI fragment (0.1 pmol; (33)). and 20 μg of extract were incubated in ligation buffer (final volume, 20 μL), for 120 min at 25° C. either in the presence or absence of ligase inhibitors (100 μmol/L). DNA fragments were resolved by separation through a 0.8% agarose gel. Labeled DNA fragments were detected in the dried gel by phosphorImager analysis (Molecular Dynamics).

Cell culture assays. Normal human breast epithelial MCF10A cells were grown in DMEM/F-12 media (Gibco) with 20 μg/ml of EGF, 0.5 μg/ml of Hydrocortisone, 0.1 μg/ml of Cholera Toxin, 10 μg/ml of Insulin, 5% horse serum and 1% Pen/Strep. Human colon cancer HCT116 cells were grown in McCoy media (Gibco) with 10% FBS and 1% Pen/Strep. Human cervical cancer HeLa and breast cancer MCF7 cells were grown in low glucose medium (Gibco) containing 10% FBS and 1% Pen/Strep.

For survival assays, cells were plated in 6 well plates and grown either in the absence or presence of ligase inhibitors. Where indicated, either MMS (0-50 μM) was added to the medium or the cells were irradiated in a Cs-137 irradiator (0-2.5 Gy). After two weeks, colonies were stained with crystal violet (Sigma) and then counted.

FACS Analysis.

Cells (1×10⁶) were serum starved for 4 days prior stimulation with serum containing medium either with or without ligase inhibitors. At various time intervals, cells were trypsinized, washed with PBS and then fixed in 95% ethanol overnight at −20° C. After RNAse treatment and propidium iodide (Sigma) staining, the cell cycle distribution was determined by FACS in the Flow Cytometry Shared Service of the Marlene and Stewart Greenebaum Cancer Center.

Cell Proliferation Assays.

The ability of the compounds identified by CADD to inhibit proliferation of normal mammary epithelial MCF10A cells and colon carcinoma HCT116 cells was determined using a Biomek FX Laboratory Automation Workstation (Beckman Coulter, Inc., Fullerton, Calif.). On day 0, 20 μl of complete medium containing the appropriate number of cells (150-300) was plated per well of a 384-well tissue plate (Fisher Scientific, Hampton, N.H.) and incubated overnight at 37° C. with 5% CO2 and 90% humidity. Next day, day 1, compounds were prepared by serial dilution with complete medium to yield the concentration 100 μM, and 20 μl was added to each well containing 20 μl of medium and cells yielding the final concentration 50 μM in 40 μl volume. Plates were incubated for additional 3 days (days-2-5) until control cells (0.5% DMSO) reached ˜70-80% confluency. On day 6, 40 μl of lysis/detection solution containing 1.2% Igepal CA-630 (Sigma) and a 1:1000 dilution of SYBR Green I nucleic acid stain (Molecular Probes, Eugene, Oreg.) was added to each well. Following an overnight incubation at 37° C., total fluorescence was measured using a Fluorostar Galaxy plate reader with a 485 nm excitation filter and 520 nm emission filter set (BMG Labtech, Inc., Durham, N.C.). Data was exported to a custom program that determined growth inhibition by dividing each individual fluorescence value by the average of fluorescence values obtained with cells treated with DMSO alone. Compounds that showed at least 40% growth inhibition compared with the DMSO-only controls inhibition of one or both of the cell lines were scored as “hits”.

The activity of hits from the initial screen was further validated using the MTT assay. Briefly, MCF10A and HCT116 cells were seeded in 96 well plates at 300 and 1200 cells per well, respectively, and allowed to adhere overnight. The following day, serial dilutions of compounds in media were added to the cells in a final volume 200 μl. After incubation for 5 days, MTT reagent (3-(4,5-dimthylthiazol-2-yl)-2,5-diphenyltatrazolium) was added and incubation was continued for 4 h. Formazan crystals generated by reduction of the MTT reagent in mitochondria were solubilized with isopropanol prior to the measurement of absorbance at 560 nm wavelength in a plate reader.

Immunocytochemistry.

The effect of ligase inhibitors on the subcellular distribution of tubulin was examined by fluorescence microscopy. Cells grown on the chamber slides were washed with PBS, fixed and then permeabilized on ice for 10 min in 2% formaldehyde and 0.1% Triton X-100. After washing with PBS, cells were incubated for 15 min with 1% BSA (Sigma-Aldrich) and then incubated with tubulin antibody (Invitrogen) for 2 h. Cells were washed with PBS prior to incubation with the AF488 conjugated secondary antibody (Invitrogen) for 1 h. After washing with PBS, cover slips were mounted on the slides with mounting media containing DAPI (Invitrogen). Cells were observed under a fluorescence microscope (Nikon Eclipse 80i) using 60× oil immersion lenses. Images were processed using NIS-Elements BR2.30 software.

Methods

CADD Screening.

The in silico identification of compounds with a high probability of binding to and inhibiting DNA ligase involved the following steps:

Identification of a putative ligand binding site on the interface between the DBD and bound DNA (FIG. 10);

molecular dynamics (MD) simulations for the generation of multiple protein conformations to address the flexibility of the binding site in the screening process;

preliminary screening of compound set,

secondary docking of compounds from the preliminary screen against the crystal structure and the MD generated structures, and

final selection of compounds for experimental assay.

Protein Structure Preparation.

The crystal structure of hLig1, obtained from the protein database bank (38) (PDB, http://www.rcsb.org/) (PDB identifier 1×9n)6 was truncated, keeping only the DBD. Hydrogen atoms were then added followed by local energy minimization with the program CHARMM (39). The minimization involved 100 conjugate gradient (CONJ) steps with the positions of atoms identified in the crystallographic structure fixed at their experimental values. Calculations were performed using the CHARMM all-atom protein force field including the CMAP modification (40, 41) with default cutoffs for the non-bond interactions. The resulting DBD structure was used in the preliminary docking (see below).

To obtain multiple conformations of the protein for secondary docking, an MD simulation was performed for 5 ns on the DBD using stochastic boundary conditions (42). First, the structure was minimized for 200 Steepest Descent (43) (SD) steps in vacuum. The binding region was then solvated by overlaying the protein with a 35 Å water sphere centered on the geometric center of the three residues, His337, Arg449, and Gly453, defining the binding pocket (see below). Water molecules within 2.8 Å to any protein non-hydrogen atom were removed. A test equilibration MD simulation showed a tendency for the water to move towards the protein and away from the surface of the sphere associated with the deletion of water overlapping the protein. Therefore, the water ball was assigned a boundary potential of 30 Å for all remaining calculations. All atoms were divided into three radial shells, i.e. the central region, an edge region from 26 to 30 Å, and an outer region beyond 30 Å, which was comprised of only protein atoms. Atoms in the outer region were constrained to their energy-minimized positions, atoms in the edge region were harmonically restrained with a force constant of 5 kcal/mol/Å and the central region was not subject to any type of restraints. The density of the water sphere was maintained using a quartic potential via the Miscellaneous Mean Field Potential (MMFP) module (44, 45) in CHARMM. Parameters defining the potential were force 0.25, droff 28.5, and pl 2.25, which yields a local well of −0.31 kcal/mol at the edge of the sphere. Non-bond interaction lists were heuristically updated out to 14 Å with the electrostatic and Lennard Jones (LJ) interactions truncated at the range of 10 to 12 Å using force switching (46). Following a 500 step SD minimization the protein was subjected to a 5 ns MD simulation at 300 K using the velocity Verlet (VVER) (47) integrator, an integration time step of 2 fs, and SHAKE to constrain all covalent bonds involving hydrogen atoms (48). Coordinates were saved every 5 ps, yielding a total of 1000 conformations from which additional structures were selected for the secondary docking. Selection of conformations for docking was performed via clustering based on pairwise root-mean square differences of the position of residues defining the binding site, i.e. the residues Glu300-Arg305 on the loop between helixes 3 and 4 according to the helix order in 1×9n.pdb, Ser334-His337 at the end of helix 5, Pro341-Asp351 on the loop following the short helix 6, and residues Gly448-Glu456 on the loop between helixes 12 and 13. Clustering was performed with NMRCLUST (49) with representative structures from the four biggest clusters chosen and used in the secondary docking.

Identification of Putative Binding Site.

A putative DNA binding site within the DBD was identified using the sphere sets used in the program DOCK (36) in combination with residues implicated in DNA binding by x-ray crystallography.

Three residues focused on, His337, Arg449, and Gly453, that are located in the central region of the DBD and make direct contacts with the DNA substrate are shown in FIG. 10. Other residues comprising the binding site include Gly448, Arg451 and Ala455.

Generation of the sphere set used the minimized crystallographic structure, with the hydrogen atoms deleted, to compute the Connolly solvent accessible surface (50, 51) via the subroutine DMS which is implemented in the program MIDAS (52) (recently updated to Chimera (53). The solvent accessible surface was computed via DMS using the surface points of the probe sphere as required when hydrogen atoms are not present, with the density of points set to 0.5 as suggested for proteins. Second, spheres ranging from radii 1.2 Å to 4 Å, complementary to the protein surface were generated by the subroutine SPHGEN implemented in the package DOCK (36). Each sphere contacts two protein surface points and lies on the normal of one of the two points. This procedure generates a very large number of spheres, which are filtered by selecting only the largest sphere associated with each surface atom. Next, spheres within 8 Å of all three reference residues, His337, Arg449, and Gly453, were selected and selected spheres on the periphery of the putative binding pocket manually deleted. This yielded the final sphere set shown in FIG. 10 which was used to direct the in silico database screening.

In Silico Compound Databases.

An in-house database of more than 4.3 million low-molecular weight compounds has been developed in the University of Maryland Computer-Aided Drug Design Center. This database is comprised of three types of files, i.e. 2D SD format files originally from the commercial vendors, 3D MOL2 format files for docking, and binary MDB format files for use in the program MOE (Chemical Computing Group Inc. Montreal, Canada). Compound preparation included removal of the smaller components in entries containing salts (e.g. counter ions), adding hydrogen atoms, assignment of the protonation state, geometry optimization using the MMFF94 (54, 55) force field level with either the SYBYL (Tripos Associates, St. Louis, Mo.) or MOE (Chemical Computing Group, Canada), and assignment of atomic partial charges based on CM2 charge model computed at the semi-empirical quantum chemical AM1 level using AMSOL (56, 57). Preliminarily screening used approximately 1,500,000 compounds from vendors chosen based on their reliability with respect to availability of compounds. Vendors include Chembridge (371,000), Chemdiv (750,000), Maybridge (60,000), MDD (33,000), Nanosyn (47,000), Specs (232,000), Timtec (165,000) and Tripos (80,000), where the values in parentheses represent the approximate number of compounds associated with each company. Recently, the compounds in the collections from these companies have been shown to typically have drug like characteristics (58).

Docking and Final Compound Selection.

Docking computations were performed using DOCK4.0 (34) with parameters previously used in our laboratory (37, 59, 60). Kollman partial atomic charges for the DBD were assigned using the program SYBYL. Database searching in DOCK is performed via a fragment-based build up procedure (61). In this approach one or more anchor fragments (e.g. rigid units, such as rings, with 5 or more atoms) are overlaid on the spheres in 200 orientations. The remainder of the ligand is then built layer by layer, with a rotation about each added bond in 10° increments to identify the most favorable orientation based on the total ligand-protein interaction energy. Thus, the docking procedure accounts for ligand flexibility while the protein is treated as rigid. From the preliminary docking using only a single conformation of the protein, the top 50,000 compounds were selected based on the normalized van der Waals attractive (Va) energy, as described below. These compounds are then subjected to a second round of docking where the crystallographic plus four additional conformations of the protein from the MD simulation (Table 1) were targeted to account for protein flexibility. The ligands were separately docked into each protein conformation, with the most favorable score from all five conformations assigned to rank that ligand. The score used in the second docking run is the total interaction energy including electrostatic and van der Waals interactions. In addition, the ligand was subjected to additional optimization by increasing the maximum anchor fragment orientations from 200 to 500, performing minimization of the anchor at each cycle of ligand buildup and minimizing the 5 inner layers upon addition of each layer of the ligand.

TABLE 1 The RMSD values in Å between each pair of the five conformations used for database screening, including the crystal structure (1 × 9n) and the four MD generated conformations (C2-C5). Only residues related to the binding region are used in the calculation. 1 × 9n C2 C3 C4 C5 1 × 9n 0 C2 2.18 0 C3 2.29 1.82 0 C4 2.23 1.65 1.42 0 C5 2.45 2.15 1.74 1.43 0

Compound clustering was performed using the Tanimoto similarity index (62, 63) based on BIT_MACCS fingerprints (64) which is implemented in the MOE software package. The BIT_MACCS fingerprints are used to compute the pairwise Tanimoto similarity matrix which contains the similarity metric between the molecular fingerprints of compounds i and j. The matrix element S(i,j), i.e. the Tanimoto coefficient (Tc) is defined as Tc=c(i,j)/u(i,j), where c(i,j) is the number of common features in the fingerprints of molecule i and j, and u(i,j) is the number of all features in the union of the fingerprints of molecule i and j (65). Two compounds are regarded as similar if S(i,j) is equal to or greater than a predefined similarity threshold. Then, from matrix S, another binary matrix O is created where each matrix element O(i,j) has the value 1 if S(i,j) is equal to or greater than the predefined similarity threshold, or 0 otherwise. Two molecules i and j are then grouped into a cluster if the overlap between the two row vectors O(i) and O(j) is greater than or equal to a predefined overlap threshold. In the present work a similarity threshold of 70% and an overlap threshold of 40% were used.

Results

In Silico Database Screening.

A putative DNA binding pocket within the DBD of hLigI was chosen as the target for a multi-tiered in silico database screening procedure, based on regions of the DBD in direct contact with the DNA in the X-ray structure of hLigI complexed with nicked DNA (FIG. 10). In the first step of the screen to identify compounds with a high probability of binding to the DBD of hLig1, ligand posing of 1.5 million compounds was based on the total interaction energy between the ligands and the protein, with ligand ranking performed using the normalized van der Waals attractive (Va) energy. Use of the Va energy parameter selects for compounds with significant steric overlap with the binding pocket and avoids compounds with highly favorable electrostatic interactions that do not fit well into the pocket. In addition, normalization procedures correct for the tendency of compound selection based on interaction energies to bias towards high molecular weight (MW) compounds (66).

Distributions of MW using different normalization procedures and the distributions of normalized scores are shown in FIGS. 11A and 11B, respectively. Based on N2/3 normalization, a total of 50,000 compounds with a molecular weight distribution centered around 300 Daltons were selected for further analysis.

Secondary screening of the 50,000 compounds applied additional energy minimization during docking and partially addressed protein flexibility (67, 68) via the inclusion of four additional, structurally diverse conformations obtained from an MD simulation. Overall, the five conformations of the DBD are similar (FIG. 12A), indicating that significant structural changes in the protein did not occur during the MD simulation. However, a detailed comparison of the orientation of the residues lining the binding region shows that there is significant diversity across the five conformations. In FIG. 12B, the orientation of three residues, His337, Arg449, and Gly453, located in the central site of the binding region are shown. Table 1 gives the root mean square deviations (RMSD) of the residues in binding region, including residues Glu300-Arg305, Ser334-His337, Pro341-Asp351, and Gly448-Glu456. Differences in the RMSD values between the crystal structure and the four conformations from MD simulation range from 1.4 to 2.5 Å, indicating significant conformational variation in the binding pocket.

Compounds were ranked based on the most favorable normalized total interaction energy of each ligand against the five protein conformations. At this stage the total interaction energy includes electrostatic interactions as well as steric considerations in the selection process. The MW and energy distributions for different powers of N normalization of the 1000 compounds with the most favorable normalized total interaction energies are shown in FIGS. 13A and 13B. These compounds were selected based on E/(N**(2/5)) normalization, yielding a MW distribution consistent with compounds known to be pharmacologically active or used in lead compound optimization studies. (69, 70)

Final selection of compounds for in vitro biochemical assays were chosen to maximize chemical diversity and based on their drug or lead-like compound properties (69, 70). Diversity was maximized by clustering the compounds based on chemical fingerprints using the Tanimoto similarity index. This yielded approximately 200 clusters of chemically similar compounds, with 1 or 2 compounds from each cluster selected based on drug- or lead-like compound properties as defined by Lipinski's rule of 5. (71) These rules include molecular weight (MW<500), adequate solubility expressed by the octanol/water partition coefficient (−5<log P(o/w)<5), number of hydrogen bond acceptors (H-bond acceptors<10), number of hydrogen bond donors (H-bond donors<5), number of rotatable single bonds (Rotatable bonds<10), and number of rings (Ring<5). The final compounds typically also satisfy the slightly stricter rules of Oprea (69). However, for clusters in which the criteria were not met, compounds were still selected for experimental assay. From this process, 233 compounds were selected for experimental testing (Table 2). Distributions of the physical and molecular properties of the 233 compounds are presented in FIG. 14, showing them to indeed fulfill Lipinski's Rule of 5.

The method for screening compounds comprises testing the compound for the ability to inhibit human DNA ligases I, III and IV with a high throughput fluorescence-based ligation assay. In this assay, phosphodiester bond formation covalently links together two oligonucleotides, one of which is a fluorescent donor AF488 that is 11 nucleotides from the 3′ terminus and the other contains a fluorescence acceptor and quencher, BHQ1 that is 15 nucleotides from the 5′ terminus. When these oligonucleotides are present in separate duplexes and in the same nicked or intact duplex, there is no significant quenching of fluorescence because the donor and acceptor are too far apart. As single-strands neither the AF488 oligonucleotide nor the BHQ1 oligonucleotides have significant secondary structure. However, when these oligonucleotides are ligated together, the resultant single strand forms a stable hairpin structure, in which the AF488 and BHQ1 groups are very close together, resulting in efficient intra-molecular quenching of fluorescence. Thus, in the DNA joining assay, the DNA substrate is denatured after incubation with the DNA ligase and then renatured in the presence of a 20-fold excess of an oligonucleotide that is identical to the ligated strand containing the fluorescent door and acceptor except that it lacks these modifications. Using this assay, DNA joining, which results in reduced fluorescence at 518 nm, can be measured rapidly, quantitatively and with high-throughput using a fluorescence plate reader.

Experimental Assays.

192 out of the 233 compounds were screened for activity in high throughput in vitro DNA ligase assays and cell culture assays (Table 3). The screens, described in detail below, were carried out in parallel.

(i) In Vitro DNA Ligase Assay.

Applicants developed a novel high throughput fluorescence-based ligation assay by modifying the sequence of the oligos to optimize hairpin formation by the single strand ligated product of the high throughput fluorescence-based ligation assay of Chen et al (22). The ability of the 192 compounds to inhibit human DNA ligases was tested. Compound stock solutions, which were 10 mM in 0.5% DMSO, were diluted in 0.5% DMSO. Human DNA ligases I and III were assayed with a nicked DNA substrate whereas as the substrate for human DNA ligase IV was two oligonucleotide duplexes with short complementary single strand ends.

In the fluorescence-based high throughput ligation assay, 15 of the 192 compounds inhibited hLigI by >50%.

In addition, to identify compounds that may be non-specific inhibitors of DNA joining, the 192 compounds were also assayed for their ability to inhibit bacteriophage T4 DNA ligase, an enzyme that utilizes the same reaction mechanism as hLigI, has similar adenylation and OB-fold domains but lacks a DBD domain. (6) Although this is also an ATP-dependent DNA ligase, it lacks the domain containing the binding pocket targeted by the in silico screen. Compounds that inhibited T4 DNA ligase were eliminated as non-specific inhibitors that most likely bind to the DNA. The results of the screen are shown in Tables 4, 5, 5A and 6.

Of the 15 compounds that inhibited hLigI (25, 32, 64, 67, 82, 113, 123, 175, 180, 189, 190, 192, 197, 200 and 202) (Table 4), 6 were active against T4 DNA ligase by >50%. Thus, the in silico screen yielded 9 compounds that specifically inhibit hLigI, a hit rate of about 5%.

One mechanism by which a compound may non-specifically inhibit human DNA ligase is by binding to DNA rather than the ligase thereby interfering with the enzyme-substrate interaction. In accord with this idea, 192, which inhibits both hLigI and T4 DNA ligase (Table 4), reduced DNA binding of the DNA intercalating agent ethidium bromide (FIG. 6B) whereas three other compounds, 67, 82 and 189 that inhibit hLigI but not T4 DNA ligase (Table 4)), had no effect on DNA binding by ethidium bromide (FIG. 6A) indicating that 67, 82 and 189 do not bind DNA.

Because the DNA binding domains of human DNA ligases III and IV and closely related to the DNA binding domain of human DNA ligase I that was used for the in silico screening, the 192 compounds identified by the in silico screening were also assayed for activity against human DNA ligases III and IV.

The in vitro screen identified compounds that are specific for each of one the human DNA ligases (DNA ligase I, DNA ligase III and DNA ligase IV) and identified compounds that inhibit two or more of the enzymes. The in vitro screen yielded three compounds that are specific for DNA ligase I (Compounds 1, 43, 82, 151, 184, 190), one compound that is specific for DNA ligase III (Compound 209), three compounds that are specific for DNA ligase IV (Compounds 93, 122, 215), four compounds that specifically inhibit DNA ligases I and III (Compound 25, 67, 200, 213), one compound that specifically inhibits both DNA ligases I and IV (Compound 113) and three compounds that inhibit all three human DNA ligases (Compound 64, 189, 197). See Table 5A for the In Vitro and In Vivo properties of the above grouped human DNA ligase inhibitors.

(ii) In Vivo Cell Culture Assays.

The 192 human DNA ligase inhibitors were screened in parallel for effects on proliferation and the ability to potentiate the effects of several DNA damaging agents using two cell human lines, a colon cancer line HCT116 and a normal breast epithelial cell line MCF10A using a Biomek FX Laboratory Automation Workstation as described (23). The DNA damaging agents used, methyl methanesulfonate (MMS), camptothecin, cis-platinum, 3 aminobenzamide and ionizing radiation, are representatives of classes of agents used to treat cancer. Briefly, HCT116 and MCF10A cells were plated in 96 well plates such that the cultures were about 80% confluent after 5 days incubation. Compounds were added to the cultures at either 15 μM or 50 μM 1 day after plating. After incubation for 3 days, cell lysed in a solution containing 1.2% Igepal CA-630 and SYBR green that stains DNA (1:1000, Molecular Probes, Eugene, Oreg.). Subsequently, fluorescence was measured per well. The studies with DNA damaging agents were performed as follows. Cells were pre-incubated with the compounds at 75 μM for 1 hour prior to the addition of camptothecin (2 nM), cis platinum (1 μM), 3 AB (2 mM) or MMS (100 μM) that resulted in dilution of the compound to a final concentration of 50 μM. For ionizing radiation, cells were pre-incubated with the compounds at 50 μM for 1 hour prior to exposure to 2 gray of ionizing radiation. Cultures were incubated for 3 days and then treated as above. The results of the in vivo cell culture assays are summarized in Table 6.

Among the 192 compounds, seven inhibit proliferation of one or both cell lines with an IC₅₀<20 μM (Compounds 16, 67, 78, 151, 165, 180, 195). At 50 μM 16 compounds inhibited proliferation of one or both cell lines by more than 40%. There was considerable overlap with the compounds identified as in vitro inhibitors of human DNA ligases. Notably, the nine of the in vitro inhibitors (compounds 43, 64, 67, 82, 151, 184, 189, 190, and 213) also inhibited cell proliferation.

In addition, three compounds act as radiosensitizers (Compounds 64, 151, 105), three compounds enhance the cytostatic effect of MMS (Compounds 67, 78, 151) and two compounds act synergistically with PARP inhibitors (Compound 64, 67).

Based on the combined results of the in vitro and in vivo screens, compounds 1, 25, 43, 64, 67, 78, 82, 93, 105, 113, 122, 151, 180, 184, 189, 197, 209, 213 and 215 were chosen for further analysis.

The specificity and inhibitory effects of a subset of these compounds were quantitated using the fluorescence-based ligation assay (22) and in gel-based assays (24).

The effect of Compounds 67, 82 and 189 on DNA joining by human DNA ligases I, III and IV and T4 DNA ligase was determined. The IC₅₀ values determined for Compounds 67, 82 and 189 using the fluorescence-based DNA joining assay (22) are shown for each of the DNA ligases. See FIG. 2A. The effect of Compounds 67, 82 and 189 on DNA joining by human DNA ligases I, III and IV and T4 DNA ligase was determined using the radioactive gel-based assay (24). See FIG. 2B. Representative gels of DNA ligation assays. The results of three independent experiments are shown graphically. For clarity, the data for T4 DNA ligase, which was not significantly inhibited, has been omitted (hLig1, □, hLigIIIβ, ∘; hLigIV/XRCC4, upside down triangle).

In addition, the mechanism of inhibition was determined. DNA ligases utilize a three step reaction to catalyze phosphodiester bond formation. In the first step, human DNA ligases interact with ATP to form a covalent enzyme-adenylate complex. When this intermediate reacts with a DNA nick, the AMP group is transferred to the 5′ phosphate terminus of the nick, forming a DNA-adenylate intermediate. Finally, non-adenylated DNA ligase catalyzes phosphodiester bond formation in a reaction that is dependent upon the hydroxyl group at the 3′ terminus of the nick and results in release of the AMP group (6).

Human DNA ligases I, III and IV and T4 DNA ligase were incubated with [α³²P] ATP in the absence or presence of compounds 67, 82 and 189 (100 μM). After separation by SDS-PAGE, the labeled ligase-AMP complex was detected by phosphorimaging.

Compounds 67, 82 and 189 did not inhibit the first step of the ligation reaction, which is DNA independent (FIG. 3A).

Since the final two steps of the ligation reaction involve interactions with DNA, we examined the effects of the ligase inhibitors on the second (FIG. 3B) (The labeled ligase-adenylate form of human DNA ligases I, III and IV and T4 DNA ligase were incubated with a linear DNA substrate containing a single non-ligatable nick in the absence or presence of compounds 67, 82 and 189 (100 μM). After separation by denaturing gel electrophoresis, the labeled DNA-AMP complex was detected by phosphorimaging (26)) and third steps (FIG. 3C) of the ligation reaction using established assays (25,26) (Non-adenylated human DNA ligases I, III and IV and T4 DNA ligase were incubated with labeled DNA-AMP in the absence or presence of compounds 67, 82 and 189 (100 μM). After separation by denaturing gel electrophoresis, labeled ligated DNA was detected by phosphorimaging (25)).

Based on these assays, we concluded that Compound 189 inhibits step 2 and Compounds 76 and 82 and inhibit step 3.

Next we used published cell extract assays (16,27,28) that measure different DNA replication and repair transactions to confirm the specificity of the DNA ligase inhibitors. FIG. 4A, the flap substrate (0.1 pmol) was incubated with cell extract (20 μg) in the absence (lane 2, Mock) or presence of 100 μmol/L, of L189 (lane 3), L67 (lane 4), or L82 (lane 5). hLigI (lane 6, I-dp) and hLigIIIa (lane 7, III-dp) were immunodepleted from the cell extracts prior to incubation with the DNA substrate. Lane 1, DNA substrate alone (Sub). The positions of the DNA substrate (24-mer), cleaved product (18-mer), and fully repaired product (43-mer). The flap substrate shown in FIG. 4A corresponds to an intermediate generated on the lagging strand during DNA replication and in the long patch subpathway of base excision repair. Previously it has been shown that DNA ligase I completes these two DNA transactions after removal of the flap by FEN-1 (16,28). A cell extract from the human cervical cancer cell line HeLa was incubated with the indicated labeled flap substrate that mimics a common intermediate in DNA replication and long patch base excision repair in the absence or presence of Compounds 67, 82, 184 and 189 (25 μM). After separation by denaturing gel electrophoresis, labeled fragments corresponding to the DNA substrate, cleaved product and fully repaired product were detected by phosphorimaging (28). Compounds 67, 82, and 189, all of which inhibit DNA ligase 1, inhibited the final ligation step of the repair reaction but had no effect on flap removal by FEN-1.

A natural AP site within a linear DNA molecule (FIG. 4B) is repaired primarily by short patch base excision repair that is completed by DNA ligase 111 (15). FIG. 4B, the linear DNA substrate with an incised AP site (0.3 pmol) was incubated with a cell extract (20 μg) and [a32P]dTTP in the absence (lane 1, Mock) or presence of 100 μmol/L of L189 (lane 2), L67 (lane 3), or L82 (lane 4). hLigI (lane 5, I-dp) and hLigIIIα (lane 6, III-dp) were immunodepleted from the cell extracts prior to incubation with the DNA substrate. The positions of the single nucleotide insertion reaction intermediate (31-mer, Incorporated) and the ligated product (73-mer, Repaired) are indicated. Thus, a HeLa cell was incubated with labeled dTTP and the indicated linear substrate with an incised AP site that mimics an intermediate in short path base excision repair in the absence or presence of Compounds 67, 82 and 189 (25 μM). Lane 6, DNA ligase I was immunodepleted from the extract prior to the assay. Lane 7, DNA ligase III was immunodepleted from the extract prior to the assay. After separation by denaturing gel electrophoresis, labeled fragments corresponding to a single nucleotide insertion and fully repaired product were detected by phosphorimaging (16). Accordingly, immunodepletion of DNA ligase III but not DNA ligase I inhibited the repair reaction (FIG. 4B). Compounds 67 and 189, each of which inhibits DNA ligase III, reduce repair of the natural AP site whereas Compound 82, which only inhibits DNA ligase I, has no effect (FIG. 4B).

Linear DNA molecules with short complementary single strand overhangs are repaired by non-homologous end joining that is completed by DNA ligase IV (27). A HeLa extract was incubated with a labeled linear cohesive-ended 1 kb fragment with cohesive ends repair in the absence or presence of Compounds 67, 82 and 189 (25 μM).). Lane 7, DNA ligase I was immunodepleted from the extract prior to the assay. Lane 8, DNA ligase III was immunodepleted from the extract prior to the assay. Lane 8, DNA ligase IV was immunodepleted from the extract prior to the assay. After separation by native agarose gel electrophoresis, the linear DNA substrate, re-circularized substrate and dimers trimers etc of the 1 kb substrate were detected by phosphorimaging (27). Accordingly, immunodepletion of DNA ligase IV but not DNA ligase I and III markedly inhibited the repair reaction (FIG. 4C) Compound 189, which inhibits DNA ligase IV, reduced joining of the linear DNA molecules whereas Compounds 67 and 82, which do not inhibit DNA ligase IV, had no effect (FIG. 4C). (Specifically, C, a 1 kb fragment with cohesive ends (0.1 pmol) was incubated with cell extract (20 μg) in the absence (lane 3, Mock) or presence of 100 μmol/L of L189 (lane 4), L67 (lane 5), or L82 (lane 6). hLig1 (lane 7, I-dp), hLigIIIa (lane 8, III-dp), and hLigIV (lane 9, IV-dp) were immunodepleted from the cell extracts prior to incubation with the DNA substrate. Lane 1, molecular mass standard (M). Lane 2, DNA substrate alone (Sub). The positions of the DNA substrate and dimmers and multimers of the substrate are indicated.)

To extend the results of the in vivo cell culture screening assay, a subset of compounds were tested in larger cultures. Different concentrations of Compounds 67 and 151 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells. Asynchronous subconfluent populations of HCT116 and MCF10A cells were pre-treated with different concentrations of compound 64 prior to the addition of 3 aminobenzamide (2 mM) MMS (100 μM) or cis-platinum (1 μM). After incubation for 5 days, cell growth was measured by the MTT assay (16).

An example of the growth inhibitory effects of Compounds 67 and 151 on the MCF10A and HCT116 cell lines is shown in FIG. 5A. Compound 64 markedly potentiates the growth inhibitory effect of the PARP inhibitor, 3 aminobenzamide on MCF10A cells (FIG. 5B).

To confirm that the DNA ligase inhibitors directly kill cells and potentiate cell killing by DNA damaging agents, we performed colony forming assays.

Different concentrations of Compound 184 were added to asynchronous subconfluent populations of HCT116, MCF7 and MCF10A cells. Colonies were counted after two weeks. (FIG. 6A).

Different concentrations of Compound 151 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells in the absence or presence of MMS (50 μM). Colonies were counted after two weeks. (FIG. 6B).

Different concentrations of Compound 189 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells. After 1 hour, cultures were irradiated with 2 gray of ionizing radiation. Colonies were counted after two weeks. (FIG. 6C).

Different concentrations of Compound 67 were added to asynchronous subconfluent populations of HCT116, MCF7 and MCF10A cells. After 1 hour, cultures were irradiated with 2 gray of ionizing radiation. Different concentrations of Compound 67 were added to asynchronous subconfluent populations of HCT116 and MCF10A cells in the absence or presence of MMS (50 μM). Colonies were counted after two weeks. (FIG. 6D).

Compound 184 kills normal breast epithelial cells (MCF10A). It is also more effective at killing colon (HCT116) and breast cancer (MCF7) cell lines (FIG. 6A). Compound 151 markedly potentiates the killing of the colon cancer cell line HCT116 by MMS but has no significant effect on MMS cytotoxicity of the normal breast epithelial cell line MCF10A (FIG. 6B). Compound 189 markedly potentiates the killing of the colon cancer cell line HCT116 by ionizing radiation but has no significant effect on the MMS cytotoxicity of the normal breast epithelial cell line MCF10A (FIG. 6C). Compound 67 also potentiates the killing of the colon cancer cell line HCT116 by ionizing radiation but has no significant effect on the MMS cytotoxicity of the normal breast epithelial cell line MCF10A (FIG. 6D, left panel). Furthermore, Compound 67 potentiates the killing of the breast cancer cell line MCF7 by both MMS and ionizing radiation but has no significant effect on the MMS- and ionizing radiation-induced cytotoxicity the normal breast epithelial cell line MCF10A (FIG. 6D, right panel). Thus, DNA ligase inhibitors are cytotoxic, enhance the cytotoxicity of DNA damaging agents and are more cytotoxic to cancer cells than normal cells.

CADD Analysis of Active Compounds.

Structures of the 10 hLigI specific inhibitors are shown in FIG. 7. As may be seen, the structures are chemical diverse, as verified by the calculation of pairwise Tanimoto similarity indices between a number of active compounds (Table 7). The largest Tc value between two compounds is 69% and the majority of the values are less than 50%, indicating a low degree of similarity between compounds. Previous studies have indicated that a value of 85% or more is associated with compounds that will have similar biological activities (72). The inclusion of chemical diversity in compound selection has the desirable effect of identify structurally dissimilar compounds for drug optimization, thereby increasing the probability of identifying active compounds. This may be seen by analyzing the energy scores of the selected compounds. Presented in FIG. 16 are the distributions of the normalized total energy scores for the 1000 compounds from the secondary screen, for the 233 compounds selected from the top 1000 based on diversity and physical properties and of 9 active compounds (FIG. 1). Consideration of diversity and physical properties led to the selection of more compounds with less favorable interaction energies. Notably, many of the active compounds would not have been selected if the top 233 scoring compounds were selected based on interaction energies and so would not have been identified.

The importance of the inclusion of multiple conformations of the putative binding site from the MD simulation in the in silico screen may be determined by simply identifying the conformation from which 10 active compounds were selected. Of the 9 hLigI specific inhibitors, one (67) was based on the crystal conformation, none were based on MD conformation C2 (time point 2.015 ns of the simulation), two (82, 113) were based on MD conformation C3 (time point 2.335 ns of the simulation), three (25, 190, 197) were based on MD conformation C4 (time point 2.950 ns of the simulation) and three (64, 189, 200) on conformation C5 (time point 3.795 ns of the simulation). Thus, the inclusion of multiple conformations is leading to the identification of additional active compounds, emphasizing the utility of this component of the screening procedure.

The protein conformation is that from the crystallographic study with the orientation of the compounds extracted from the individual conformations following alignment of the protein conformations as shown in FIG. 12A. All the inhibitors occupy the targeted site, consistent with the docking methodology. However, they do sample different regions of the binding site. Such difference may contribute to differential selectivities of activity of the inhibitors for different ligases.

Three of the active compounds, 67, 82, and 189 have been subjected to more extensive biological characterization (73). Although all three compounds are predicted to bind in the putative binding site, they do exhibit some level of variability in the binding orientations. Interestingly, while all three compounds inhibit hLigI but not T4 DNA ligase (Table 4), their activity versus the other human DNA ligases differs significantly. While 82 inhibits only hLigI, 67 inhibits both hLigI and hLigIII and 189 inhibits all three human DNA ligases. Presumably, differences in the specificities of the inhibitors for the three human DNA ligases reflect a combination of differences in the binding modes of the structurally diverse inhibitors and differences in the molecular architecture of the targeted DNA binding pocket between the three human DNA ligases. Importantly, inhibitors with defined specificities for the different human DNA ligases will be invaluable reagents for elucidating the physiological roles of human DNA ligases.

Consistent with the inclusion of physical properties in the selection process, all the hLigI specific inhibitors fall into the drug-like range according to Lipinski's rule of five (Table 4) (71), while still spanning a range of physical properties.

From the in silico database Applicants have identified compounds of General Formulae I-XVI as also being DNA ligase inhibitor candidates.

General Formulae I and II are illustrative of compounds related to Compound 64. Compounds related to Compound 64 are shown in Table 8.

General Formulae IIA and IIIA are illustrative of compounds related to Compound 67. Compounds related to Compound 67 are shown in Table 9.

General Formulae III is illustrative of compounds related to Compound 78.

Compounds related to Compound 7$ are shown in Table 10.

General Formulae IV and V are illustrative of compounds related to Compound 113. Compounds related to Compound 113 are shown in Table 11.

General Formulae IX and X are illustrative of compounds related to Compound 151. Compounds related to Compound 151 are shown in Table 12.

General Formulae XI and XII are illustrative of compounds related to Compound 180. Compounds related to Compound 180 are shown in Table 13.

General Formulae XIII and XIV are illustrative of compounds related to Compound 189. Compounds related to Compound 189 are shown in Table 14.

General Formulae XV and XVI are illustrative of compounds related to Compound 197. Compounds related to Compound 197 are shown in Table 15.

where R₂ is hydrogen, R₃ is hydrogen, halogen, Cl,

(where R₄ is halogen or Cl and n is 0-5), or

R₁ is

(where R₆ is halogen or F and n is 0-5), or

(where R₆ is halogen or F and n is 0-5)

where R₁, R₂ and R₃ are the same as in formula (I)

R₂ are each the same or different and are selected from —OH, alkoxy, halogen, —CH₃, —OCH₃ Cl, Br and F or two R₂s form a fused ring and n=0-3, preferably 0-2 R₃=H, alkyl, or —CH₃ R₄=H, alkyl, methyl or ethyl; or one of R₃ and one of R₂ form a fused ring R₁=aryl,

(where R₅ is halogen, alkyl, Br, Cl, or —CH₃ and n₅ is 0-5)

(where R₅ and is halogen, alkyl, Br, Cl, or —CH₃ and n₅ is 0-5) or R₁ and R₄ forma fused ring.

Where R₁, R₂, R₃, R₄, and n are the same or in general formula (IIA)

-   -   R₅ and n₅ are same as R₂ and n in general formula (IIA).

where R₁ is alkyl, alkylene, alkoxy, ethyl, methyl, alkyl substituted with aryl,

R₂ is O⁻, halogen, alkyl, alkoxy, BR, Cl, —O—CH₃, —NH₂, —CH₃, and when n is greater than 2, two R₂s may form a fused ring, n is 1-5.

wherein R₅, R_(5′) R_(5″) and R_(5′″) are selected from H, aryl, and alkyl; and preferably H wherein R₁, R_(1′) R_(1′″) and R_(1′″) are selected from

wherein each R₇ is independently selected from halogen, Cl, Br, F, alkyl, and —CH₃, and n₇ is 0-5

where R₈ and n₈ are the sane as R₇ and n₇,

where R₉ and n₉ are the same as R₇ and n₇, alkyl-cycloalkyl,

where R₁₀ and n₁₀ the same as R₇ and n₇,

where z is 1, 2, 4 or 5, R₁₀ is selected from halogen, Br and Cl, R₂₀ is selected from alkylene, cycloalkyl, alkyl-aryl,

-   -   where R₂₉, R₃₀ and R₃₁ are selected from halogen, Cl, I, alkyl     -   n₂₉, n₃₀ and n₃₁ are 0-5; preferably 0 or 1     -   n₁₀ is 0-5; preferably 0 or 1.

-   -   wherein     -   R₂ is selected from —OH, O—,

alkoxy, halogen, Cl, and —O—CH₃,

-   -   n is 0-5, preferably 1 or 2,     -   R₃ is selected from H, alkyl, and —CH₃,     -   R₁ is selected from H, alkyl, and CH₃, and     -   X is selected from N and CH.

-   -   wherein     -   R₂ is selected from —OH, O—,

alkoxy, halogen, Cl, and —O—CH₃,

-   -   X₁ is selected from N and CR₃,     -   X₂ is selected from N and CR₄,     -   wherein R₃ and R₄ are independently selected from H and

-   -   and at least one of X₁ or X₂ is

-   -   R₅ is selected from H, alkyl, methyl, halogen and Cl,     -   R₁ is selected from aryloxy, alkoxy,

where R₆ is selected from alkyl, —CH₃, H, alkoxy, —OCH₂—CH₃, halogen, Cl, Br

n₆ is 0-6;

-   -   n₁ is 0-3     -   R₂ is selected from 1-1, alkyl, halogen, methyl, and Cl, and     -   n₂ is 0 to 5.

-   -   where R₁, R₂ and n₂ are same as defined in Formula (XI).

-   -   R₁ is selected from H, alkyl, methyl, —C≡N, halogen and Cl, or         when n is greater than 2 two R_(a)s may form a fused ring     -   n is 0-5,     -   R₃ is selected from H alkyl and methyl,     -   R₂ is selected from —OH and —NH₂,     -   X is selected from N and CR₄, where R₄ is selected from —OH, N         and H,     -   R₅ is selected from H, —OH, NH₂ and S

-   -   R₁ is selected from H, alkyl, methyl, —C≡N, halogen and Cl, or         when n is greater than 2 two R₁s may form a fused ring     -   n is 0-5,     -   R₂, R₃ and R₄ are independently selected from —O and —N.

-   -   R₁ and R₂ are independently selected from

-   -   R₂ and R₄ are independently selected from alkyl, alkyl, methyl         and benzyl     -   n₁ and n₂ are independently selected from 0, 1, 2.

-   -   R₁ and R₂ are independently selected from

-   -   R₂ and R₄ are independently selected from alkyl, alkyl, methyl         and benzyl     -   n₁ and n₂ are independently selected from 0, 1, 2.

The DNA ligase inhibitors of the present invention may also be used as research tools in addition to therapeutics. As research tools, the DNA ligase inhibitors may be used to identify the DNA ligase involved in different DNA transactions either in cell culture and/or in cell extract based assays. Including the use of DNA ligase inhibitors to provide novel insights into the reaction mechanisms of human DNA ligases; the use of DNA ligase inhibitors in cell extract assays to identify the human DNA ligase involved in DNA repair pathways; and the use of DNA ligase inhibitors in cell culture assays to identify the human DNA ligase involved in different DNA repair pathways in vivo. For example, delineating DNA ligases participating and determining which of multiple pathways are being used.

The DNA ligase inhibitors of the present invention may be used to facilitate in vitro and in vivo studies delineating the cellular functions of these enzymes.

Most DNA damaging agents introduce more than one type of DNA lesion. In addition, a specific DNA lesion may be removed by more than one DNA repair pathway. Since the DNA ligase inhibitors of the present invention inhibit DNA repair pathways, the use of the inhibitors in a combination of extract and cell culture assays may be used to delineate the DNA repair pathways involved in the repair of DNA lesions introduced by a DNA damaging agent and the DNA repair pathways involved in the repair of a specific DNA lesion.

The DNA ligase inhibitors of the present invention may be used as research tools to promote the understanding of genome stability and DNA repair, such as Lagging strand DNA replication; Telomere replication; Rescue of stalled replication forks; Damage response signaling cascade; DSB repair, BER; polβ-dependent long patch; PCNA-dependent long patch; Short patch; and NER: DNA double strand break repair by homology-dependent pathways and by the major and alternative non-homologous end-joining pathways.

The DNA ligase inhibitors of the present invention may also be used in drug development for clinical cancer treatment, such as anti-cell proliferation and Radiosensitizers.

As used herein, “treat” means alter, apply, effect, improve, care for or deal with medically or surgically, ameliorate, cure, stop and/or prevent an undesired biological (pathogenic) process. The skilled artisan is aware that a treatment may or may not cure.

As used herein, the effective amount or “therapeutically effective amounts” of the compound of the present invention to be used are those amounts effective to produce beneficial results in the recipient animal or patient. Such amounts may be initially determined by reviewing the published literature, by conducting in vitro tests or by conducting metabolic studies in healthy experimental animals. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated. Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value.

A therapeutically effective amount of a compound of the present invention as a treatment varies depending upon the host treated and the particular mode of administration. The term “body weight” is applicable when an animal is being treated. When isolated cells are being treated, “body weight” as used herein means “total cell body weight”. The term “total body weight” may be used to apply to both isolated cell and animal treatment. Those of skill in the art will recognize the utility of a variety of dosage range.

TABLE 2 ligase_1x9n_ docking_233_compounds COMP_NAME IDNUMBER stru MolW logP 1

chembridge000828 5103856 2335 174 1.92 2

chembridge004647 5133963 2335 238 0.724 3

chembridge004783 5135431 1x9n 226 −0.672 4

chembridge005314 5140822 1x9n 198 0.552 5

chembridge009555 5185417 2335 281 2.54 6

chembridge0119938 6635973 1x9n 337 1.05 7

chembridge0132951 5540113 2950 295 4.32 8

chembridge0136123 5649818 2950 316 −1.87 9

chembridge0137192 5679271 2950 326 0.653 10

chembridge0144275 5807168 1x9n 322 2.65 11

chembridge014881 5226945 2950 381 4.06 12

chembridge015172 5227796 2950 232 1.72 13

chembridge0171207 6102396 2335 433 2.3 14

chembridge0171753 6106327 2950 235 0.925 15

chembridge017857 5241412 1x9n 377 1.54 16

chembridge017892 5241504 2335 300 2.36 17

chembridge0183353 6203744 2950 218 0.384 18

chembridge0192792 6364469 2950 270 2.85 19

chembridge0222418 6629596 2335 275 −0.025 20

chembridge0250465 5925673 2950 230 0.83 21

chembridge0253645 6297813 2950 210 −0.982 22

chembridge0257297 6550468 2950 294 3.43 23

chembridge0266747 6841883 2335 301 0.704 24

chembridge0295137 7173063 1x9n 356 1.02 25

chembridge029819 5317419 2950 191 0.119 26

chembridge0307818 7296187 2950 271 1.29 27

chembridge0343539 7650920 2335 308 1.64 28

chembridge0347904 7676009 1x9n 344 2.26 29

chembridge0352654 7699479 2335 383 4.76 30

chembridge0355774 7724431 2950 318 1.63 31

chembridge036048 5355751 2335 293 0.343 32

chembridge0365296 7777402 2335 386 1.98 33

chembridge0374491 7812749 2950 237 3.38 34

chembridge0374541 7813065 2335 303 1.57 35

chembridge0377382 7830700 2950 233 2.01 36

chembridge0378150 7836125 2950 357 2.28 37

chembridge0382972 7863038 2335 222 0.311 38

chembridge0387746 7902307 2335 312 −0.012 39

chembridge063174 5608638 2950 305 1.95 40

chembridge080590 5742694 2335 315 1.71 41

chembridge085143 5790780 2335 304 1.91 42

chembridge091964 5869880 2335 301 2.79 43

chembridge099119 6051018 1x9n 335 4.36 44

chembridge102405 5356872 2950 312 1.5 45

chembridge103842 5154723 2335 329 3 46

chembridge104229 5210894 2335 339 1.61 47

chemdiv015291 3448-0483 2335 276 2.97 48

chemdiv016239 3464-8119 2335 309 1.56 49

chemdiv018608 2279-5355 2950 279 1.5 50

chemdiv0202765 000A-0636 2335 225 −0.669 51

chemdiv0223644 4300-0817 2335 231 1.6 52

chemdiv0254427 K831-0255 2950 300 0.535 53

chemdiv0260892 0054-0264 2950 210 0.972 54

chemdiv0262519 1725-0122 2335 129 −0.903 55

chemdiv0281075 4055-0046 2335 205 −0.375 56

chemdiv0283584 4114-0028 2950 305 2.15 57

chemdiv0283734 4130-0051 2335 260 0.684 58

chemdiv0297199 4596-0306 1x9n 175 0.682 59

chemdiv0297579 4676-0062 2950 252 2.22 60

chemdiv0310162 8011-6595 2950 204 0.399 61

chemdiv0311450 8012-0236 2335 340 3.58 62

chemdiv0329374 K831-0247 2950 287 0.226 63

chemdiv0330961 K906-0447 2950 369 1.03 64

chemdiv0333435 R052-1644 2950 290 2.03 65

chemdiv071566 0814-0180 2950 321 3.1 66

chemdiv093992 1535-0002 2335 224 0.103 67

chemdiv102812 1761-0019 1x9n 486 4.79 68

chemdiv103836 1761-1961 1x9n 370 0.978 69

chemdiv117392 2093-0008 2950 193 0.834 70

chemdiv137918 2595-1130 2335 219 0.647 71

chemdiv172782 8007-0302 2950 320 0.727 72

chemdiv189691 8010-4604 2335 356 4.22 73

chemdiv195033 K061-1138 2950 220 0.575 74

chemdiv201905 K781-0461 2335 259 1.7 75

chemdiv202632 R095-0016 2950 306 3.66 76

chemdiv4000099 000A-0435 2950 205 0.146 77

chemdiv4000752 0519-0362 2950 331 0.823 78

chemdiv4003854 2672-0239 2335 321 2.85 79

chemdiv4003904 2684-1487 2950 309 1.16 80

chemdiv4004117 2764-0217 2950 259 0.825 81

chemdiv4006822 3350-0006 2950 266 1.75 82

chemdiv4014181 4300-0746 2335 310 4.72 83

chemdiv4016932 4469-0504 2950 375 4.66 84

chemdiv4016952 4470-0573 2950 310 0.754 85

chemdiv4017354 4483-2627 2950 321 3.86 86

chemdiv4030337 5167-1580 2335 328 2.46 87

chemdiv4032105 5227-2224 2335 219 2.2 88

chemdiv4047092 5720-0252 2335 182 −0.203 89

chemdiv4050365 5849-2464 2335 244 0.952 90

chemdiv4059074 6144-0830 2335 250 0.548 91

chemdiv4064940 6296-0075 2335 302 2.87 92

chemdiv4067503 6404-0306 2950 252 1.83 93

chemdiv4077013 7011-1829 2950 194 0.386 94

chemdiv4077303 7100-1148 2335 313 2.36 95

chemdiv4077895 7213-0775 2335 207 −1.01 96

chemdiv4078007 7287-0119 2335 399 0.835 97

chemdiv4080636 8010-0511 2950 250 2.1 98

chemdiv4080731 8010-5081 2335 189 0.518 99

chemdiv4084070 8013-6158 2335 264 0.637 100

chemdiv4088658 8015-2157 2335 180 −1.17 101

chemdiv4092369 C066-3475 2335 234 0.82 102

chemdiv4113108 C200-0872 2335 302 1.7 103

chemdiv4114091 C200-2517 2335 277 0.282 104

chemdiv4115701 C206-0731 2335 239 2.52 105

chemdiv4115760 C206-0886 2950 302 4.09 106

chemdiv4136188 C301-1637 2950 178 1.51 107

chemdiv4155887 C455-0040 2950 258 1.39 108

chemdiv4178771 C612-0733 2950 312 1.48 109

chemdiv4200659 C804-0249 2950 343 0.498 110

chemdiv4212928 E546-0207 2335 287 1.24 111

chemdiv4226558 K783-5936 2950 208 0.537 112

chemdiv4247207 K906-0577 2950 300 1.38 113

chemdiv4249937 K978-1019 2335 475 1.42 114

chemdiv4250212 R052-2665 2950 163 −0.321 115

chemdiv4250230 R052-2693 2950 174 0.921 116

chemdiv4256344 C200-2775 2950 475 0.953 117

chemdiv4257714 C430-0780 2950 227 0.861 118

chemdiv4262012 C800-0149 2950 244 1.26 119

maybridge0404348 BTB 06428 2950 247 3.81 120

maybridge0405401 BTB 08297 2950 323 1.9 121

maybridge0407919 BTB 13897 2335 207 2.38 122

maybridge0407972 BTB 14052 2950 216 1.27 123

maybridge0409092 CD 01419 2950 228 1.51 124

maybridge0414926 GK 01940 2335 237 1.86 125

maybridge0418520 HTS 03891 2950 314 2.34 126

maybridge0419334 HTS 05121 2950 234 0.154 127

maybridge0425599 JFD 00838 2950 341 1.69 128

maybridge0428829 KM 02107 2950 250 0.522 129

maybridge0430404 KM 05117 2950 207 0.114 130

maybridge0434449 MWP 00580 2950 189 1.07 131

maybridge0435308 NRB 00719 2950 289 2.87 132

maybridge0439282 RF 00190 2950 215 0.428 133

maybridge0439473 RF 00770 2950 268 2.75 134

maybridge0440186 RF 03622 2335 190 1.67 135

maybridge0441358 RH 00871 2950 239 0.994 136

maybridge0442630 RJC 00691 2335 183 0.161 137

maybridge0443763 RJC 02884 2950 244 2.06 138

maybridge0445673 S 00903 2335 258 4.11 139

maybridge0445675 S 00982 2950 227 3.37 140

maybridge0449798 SCR 01207 2950 342 2.75 141

maybridge0454605 SPB 00315 2335 167 1.14 142

maybridge0455528 SPB 02143 2950 346 2.91 143

maybridge0455775 SPB 02523 2950 198 2.39 144

mdd0506944 APX000008174 2335 282 3.44 145

mdd0507227 APX000008527 2950 254 3.16 146

mdd0507257 APX000008566 2950 256 1.56 147

mdd0525205 APX000027192 2335 179 0.51 148

mdd0528932 APX000030930 2335 203 0.857 149

mdd0531592 APX000033591 2950 317 1.52 150

nanosyn000678 NS49351 2335 361 3.55 151

nanosyn006272 NS54884 2950 306 0.108 152

nanosyn012438 NS15117 1x9n 304 4.26 153

nanosyn019560 NS0633 1x9n 296 2.12 154

nanosyn020035 NS1965 2950 202 2.14 155

nanosyn020419 NS2538 2950 347 0.517 156

nanosyn022218 NS5033 2950 224 −1.3 157

nanosyn024054 NS7425 2335 252 1.04 158

nanosyn026199 NS10673 2335 288 −0.938 159

nanosyn028817 NS14410 2335 305 2.9 160

nanosyn029397 NS15310 2335 312 2.96 161

nanosyn032970 NS21015 2335 246 1.61 162

nanosyn041421 NS32415 2950 200 −1.14 163

nanosyn042848 NS33842 2335 254 1.25 164

nanosyn043053 NS34047 2950 209 1.02 165

nanosyn044719 NS35713 1x9n 334 4.05 166

nanosyn047911 NS38906 2950 195 0.555 167

nanosyn052735 NS44149 2335 224 0.92 168

nanosyn061098 NS63350 1x9n 253 2.32 169

nanosyn064176 NS66483 2335 289 2.57 170

nanosyn064661 NS67013 2335 269 1.3 171

specs0087646 AG-205/12145002 2335 272 0.787 172

specs0093768 AP-845/40883774 2950 228 1.8 173

specs0094412 AE-641/40197985 2950 279 1.9 174

specs0094816 AI-942/25121085 2950 206 0.428 175

specs0094999 AM-807/25050007 2335 174 0.998 176

specs0096856 AE-406/41056556 2335 259 1.84 177

specs0097907 AE-641/30103046 2335 265 4.42 178

specs0104444 AF-399/15335020 2335 292 1.59 179

specs0104985 AF-399/15393031 1x9n 355 3.6 180

specs0112290 AG-205/15425118 2950 301 2.44 181

specs0123459 AG-690/09793058 2335 200 −1.54 182

specs0126485 AG-690/11384661 2950 291 −0.44 183

specs0129149 AG-690/12002297 2335 302 2.65 184

specs0133795 AG-690/15436354 2950 272 −0.45 185

specs0140784 AG-690/40697266 2335 345 2.84 186

specs0142480 AG-690/40700254 2335 324 2.14 187

specs0142745 AG-690/40700684 2335 354 0.846 188

specs0154616 AI-204/31687014 2950 216 1.29 189

specs4002175 AC-907/34129012 2950 246 1.84 190

specs4002201 AC-907/34130009 2950 270 1.54 191

specs4003032 AE-562/12222186 2950 344 0.148 192

specs4003077 AE-562/12222297 2950 274 1.56 193

specs4004319 AE-641/06280018 2950 311 3.11 194

specs4007749 AE-848/30709022 2950 306 2.11 195

specs4020536 AG-205/08231019 2335 316 3.81 196

specs4021096 AG-205/08625012 2335 273 0.787 197

specs4028351 AG-205/32243059 2950 310 1.63 198

specs4030921 AG-205/33652013 2950 292 1.63 199

specs4036585 AG-205/40650755 2950 362 1.61 200

specs4039932 AG-207/37370001 2335 214 2.8 201

specs4043054 AG-670/31548028 2950 287 0.234 202

specs4044861 AG-690/08639033 1x9n 390 4.49 203

specs4051894 AG-690/12844905 2950 204 1.82 204

specs4052603 AG-690/12885209 2950 216 1.3 205

specs4056295 AG-690/15433670 2950 205 0.928 206

specs4056597 AG-690/15438954 2950 247 0.308 207

specs4063372 AG-690/37215010 2950 289 1.38 208

specs4073894 AI-237/31666029 2335 272 0.565 209

specs4074491 AJ-030/12105064 2335 370 −0.561 210

specs4074773 AJ-087/41885602 2950 241 1.57 211

specs4076821 AJ-292/41686278 1x9n 366 2.78 212

specs4078664 AJ-333/36115017 2950 212 −0.117 213

specs4079101 AK-087/42718317 2950 194 2.54 214

specs4085187 AK-918/42409851 2950 312 3.63 215

specs4087862 AK-968/11789151 2335 297 1.28 216

specs4103227 AK-968/41169454 2335 221 2.59 217

specs4103395 AK-968/41170109 2335 219 0.442 218

specs4121778 AN-465/41521127 2950 226 1.49 219

specs4132753 AN-829/13872035 2950 196 −0.685 220

specs4133908 AN-979/15013141 2335 331 1.81 221

specs4136100 AO-080/42479361 2950 318 3.23 222

speccs4156297 AR-422/41026969 2335 315 1.36 223

st007595 st008467 2335 197 1.3 224

tripos0034247 1525-00782 2950 249 0.974 225

tripos0034504 1525-01725 2335 279 1.36 226

tripos0046768 1528-02292 2335 301 0.506 227

tripos0047216 1528-03826 2950 342 3.44 228

tripos0047548 1528-04508 2335 258 2.35 229

tripos0049773 1528-10011 2950 336 2.6 230

tripos0049952 1528-10605 2950 279 2.27 231

tripos0052524 1533-00601 1x9n 231 1.08 232

tripos0053972 1533-06492 2335 423 4.12 233

tripos0074665 1554-07198 1x9n 362 3.09

TABLE 8 ligase64 chemdiv0333435 75 COMP NAME IDNUMBER MW a d logP 1

chemdiv0333435 R052-1644 288.6 2. 0. 2.15 2

chembridge0127433 5241477 258.6 3. 2. 0.88 3

chembridge017871 5241438 333.6 2. 1. 3.11 4

chembridge0224352 6640684 384.3 4. 0. 2.35 5

chembridge0252784 6208029 363.7 2. 1. 3.65 6

chembridge0318761 7399643 252.2 2. 2. 1.85 7

chembridge0350072 7684126 345.1 1. 1. 3.81 8

chembridge0353163 7703755 284.2 1. 1. 3.16 9

chembridge0361286 7749202 295.2 3. 2. 2.81 10

chembridge0410035 7963899 441.3 5. 0. 2.20 11

chembridge057754 5556669 269.2 1. 0. 2.72 12

chemdiv0235647 8012-5585 418.8 4. 2. 3.62 13

chemdiv029346 8002-5557 216.2 1. 0. 0.99 14

chemdiv195033 K061-1138 218.5 1. 0. 0.70 15

chemdiv202374 R052-0733 254.2 2. 0. 1.52 16

chemdiv4039985 5555-0004 216.2 2. 2. 0.94 17

chemdiv4228031 K784-6223 364.3 3. 2. 3.13 18

chemdiv4230965 K786-1157 300.6 3. 1. 1.47 19

chemdiv4231246 K786-1552 408.8 3. 1. 3.72 20

maybridge0431189 KM = 06791 257.2 3. 1. 2.02 21

maybridge0431220 KM = 06833 241.2 3. 1. 1.20 22

maybridge0431223 KM = 06837 291.2 2. 0. 3.70 23

maybridge0431242 KM = 06873 363.3 3. 0. 3.61 24

maybridge0431243 KM = 06874 345.3 3. 0. 3.45 25

maybridge0431901 KM = 08121 213.1 1. 0. 1.23 26

specs0118592 AG-227/40703560 347.7 3. 1. 3.59 27

specs4043497 AG-670/36154046 359.3 2. 2. 3.64 28

specs4099247 AK-968/37166230 294.6 1. 0. 2.66

TABLE 9 ligase-67-chemdiv102812-similar90 COMP_NAME IDNUMBER Weight logP(o/w) 1

chemdiv102812 1761-0019 486.12 4.794 2

chembridge0139405 5724812 423.22 3.619 3

chembridge0163789 6046146 516.15 4.785 4

chembridge061292 5575463 407.22 3.959 5

chembridge091422 5863488 414.42 5.27 6

chemdiv0304474 8004-4208 547.36 6.79 7

chemdiv030710 8002-9168 449.26 3.468 8

chemdiv031476 8003-1205 405.3 2.642 9

chemdiv031594 8003-1363 542.3 5.168 10

chemdiv032220 8003-2671 390.78 3.338 11

chemdiv034545 8004-0876 407.22 3.961 12

chemdiv036531 8004-5914 373.32 3.096 13

chemdiv038132 8005-0790 421.21 3.278 14

chemdiv042897 8006-1915 360.3 2.672 15

chemdiv042898 8006-1916 376.76 3.074 16

chemdiv042910 8006-1930 360.3 2.672 17

chemdiv043057 8006-2113 515.31 5.569 18

chemdiv088176 1359-0042 419.23 3.514 19

chemdiv102882 1761-0255 486.12 4.794 20

chemdiv103694 1761-1563 421.25 4.704 21

chemdiv159407 3284-0676 378.77 3.45

TABLE 10 ligase 78 chemdiv4003854 83 COMP NAME IDNUNBER cl . . . MW logP 1

chemdiv4003854 2672-0239 1.00 320. 2.97 2

chembridge066517 5635200 12.0 399. 3.81 3

chembridge066825 5637177 13.0 449. 4.93 4

chembridge081742 5756943 15.0 384. 4.19 5

chembridge084110 5785385 17.0 308. 2.65 6

chemdiv030495 8002-8328 19.0 385. 4.24 7

chemdiv049350 8010-0528 16.0 358. 3.87 8

chemdiv049352 8010-0530 21.0 337. 1.94 9

chemdiv070385 0784-0310 22.0 413. 3.09 10

chemdiv070399 0784-0352 10.0 370. 4.10 11

chemdiv080501 1094-0055 30.0 319. 3.50 12

chemdiv080519 1094-0096 31.0 305. 3.16 13

chemdiv080520 1094-0097 30.0 319. 3.46 14

chemdiv080523 1094-0100 35.0 320. 2.49 15

chemdiv080527 1094-0104 1.00 320. 2.97 16

chemdiv080533 1094-0112 1.00 370. 4.27 17

chemdiv080539 1094-0122 41.0 378. 2.11 18

chemdiv085129 1270-0044 46.0 335. 3.12 19

chemdiv085130 1270-0045 47.0 385. 4.24 20

chemdiv105653 1783-0201 52.0 355. 4.29 21

chemdiv133102 2395-0012 57.0 310. 1.73 22

chemdiv4003850 2672-0163 10.0 370. 4.10 23

chemdiv4003855 2672-0240 1.00 370. 4.19 24

chemiv4019950 4555-4523 16.0 308. 2.65 25

chemdiv4021425 4632-6961 70.0 354. 3.60 26

chemdiv4021435 4632-6987 72.0 433. 4.44 27

chemdiv4021439 4632-7005 75.0 404. 4.73 28

specs0122994 AG-690/09504058 117. 414. 2.97

TABLE 11 Ligase_active#113_chemdiv4249937 COMP_NAME IDNUMBER MW logP 8585 1

113_chemdiv4249937 K978-1019 475 1.42 1 2

amb_c0025720 A2357/0099549 393 3.31 1 3

amb_c0045243 AmblieP-559829 321 0.481 3 4

amb_d022599 A2357/0099549 393 3.31 1 5

amb_e0017501 ASKBASE/24046 321 0.481 3 6

amb_e0021248 ASKBASE/28478 383 2.31 6 7

amb_e0021385 ASKBASE/28630 318 1.6 7 8

amb_e0023055 ASKBASE/30341 383 2.31 8 9

amb_e0023957 ASKBASE/31261 383 2.34 8 10

amb_e0025034 ASKBASE/32342 335 0.569 10 11

amb_e0025080 ASKBASE/32388 353 2.19 1 12

amb_e0025103 ASKBASE/32411 397 2.39 1 13

amb_e0025149 ASKBASE/32457 332 1.68 13 14

asinex59633 BAS = 0138030 638 7.73 14 15

chembridge0216275 6584322 383 2.34 8 16

chembridge0216484 6585646 383 2.31 8 17

chembridge0218325 6599829 332 1.68 13 18

chembridge0258673 6620392 353 2.19 1 19

chembridge0266367 6831135 350 1.93 7 20

chembridge0318078 7390552 338 2.4 20 21

chembridge0368775 7788404 443 4.64 1 22

chembridge0372356 7802552 423 4.14 1 23

chembridge0408597 7957540 421 4.19 1 24

chemdiv0315353 8012-8697 421 4.19 1 25

chemdiv0324687 K781-2046 367 2.68 1 26

chemdiv0324691 K781-2050 401 3.28 1 27

chemdiv0325214 K781-4155 415 2.95 27 28

chemdiv0325222 K781-4171 397 2.8 1 29

chemdiv0325224 K781-4177 446 3.48 29 30

chemdiv0325227 K781-4185 411 3.1 27 31

chemdiv0325238 K781-4295 350 2.24 1 32

chemdiv0325309 K781-4963 457 3.7 32 33

chemdiv0325311 K781-4969 457 4.28 1 34

chemdiv0325312 K781-4973 488 4.81 1 35

chemdiv0325313 K781-4974 389 3 32 36

chemdiv0325316 K781-4979 431 4.32 1 37

chemdiv0325327 K781-4981 508 5.35 1 38

chemdiv0325318 K781-4982 439 4.13 1 39

chemdiv0326079 K783-0131 465 5.31 39 40

chemdiv0328145 K784-4328 432 3.39 27 41

chemdiv4130154 C274-3672 565 2.95 41 42

chemdiv4148442 C382-0316 479 5.75 39 43

chemdiv4148450 C382-0342 451 4.86 1 44

chemdiv4225320 K781-4165 411 2.89 1 45

chemdiv4225426 K783-0142 437 4.42 39 46

chemdiv4234898 K786-6828 382 2.22 46 47

chemdiv4238958 K788-2181 435 4.24 1 48

chemdiv4239059 K788-2347 465 5.22 1 49

chemdiv4239292 K788-2747 449 4.54 1 50

chemdiv4239818 K788-3519 469 4.84 1 51

chemdiv4239820 K788-3521 487 5.34 1 52

chemdiv4248907 K938-0642 419 3.81 1 53

chemdiv4248970 K938-0786 435 4.24 1 54

comgenex029401 CGX-0399491 441 4.33 1 55

specs0087828 AG-205/41005779 421 4.19 1 56

specs4072254 AH-487/42307105 435 4.84 1 57

specs4072408 AH-487/42483123 457 5.11 1 58

specs4072415 AH-487/42483920 457 4.73 1 59

specs4072424 AH-487/42485536 457 4.73 1 60

specs4077937 AJ-292/42062402 332 1.68 13 61

specs4077961 AJ-292/42062682 383 2.34 8 62

specs4108021 AK-968/41927486 515 -0.294 62 63

specs4135786 AO-080/41280680 393 3.31 1 64

timtt031879 ST031879 383 2.31 8 65

timtt044045 ST044045 383 2.34 8 66

timtt044162 ST044162 397 2.39 1 67

timtt044167 ST044167 332 1.68 13 68

timtt045785 ST045785 421 4.19 1 69

tripos0051110 1532-03029 396 0.623 13 70

tripos0051115 1532-03050 391 2.38 13

TABLE 12 ligase 151 nanosyn006272 s85 COMP NAME IDNUMBER logP MW 1

nanosyn006272 NS54884 0.11 306. 2

chembridge0136023 5648015 -0.42 263. 3

chembridge0139327 5722560 2.90 373. 4

chembridge065828 5630287 0.46 304. 5

chembridge070734 5663902 0.45 386. 6

chembridge073257 5677048 -0.20 307. 7

chembridge074051 5681228 0.92 321. 8

chemdiv0227504 4487-0452 -0.19 277. 9

chemdiv023628 8001-0622 0.31 304. 10

chemdiv0308002 8008-5425 2.16 366. 11

chemdiv037566 8004-9459 0.31 320. 12

chemdiv085124 1270-0021 3.07 458. 13

chemdiv103764 1761-1799 2.34 366. 14

chemdiv103782 1761-1819 0.11 306. 15

chemdiv103828 1761-1952 1.01 324. 16

chemdiv103830 1761-1954 0.64 304. 17

chemdiv103836 1761-1961 1.10 368. 18

chemdiv103848 1761-1976 0.71 334. 19

chemdiv106079 1805-1308 1.21 382. 20

chemdiv108507 1897-1408 0.43 303. 21

chemdiv110779 1981-1490 1.91 427. 22

chemdiv119094 2144-0787 -0.45 336. 23

chemdiv133064 2391-2374 2.45 395. 24

chemdiv159730 3284-1651 1.84 316. 25

chemdiv197874 K089-0087 3.19 394. 26

chemdiv4107645 C163-0462 0.11 291. 27

chemdiv4107646 C163-0463 0.41 305. 28

mdd022784 st016688 3.25 409. 29

nanosyn013594 NS18706 3.40 472. 30

specs0169427 AK-968/15605425 1.77 399. 31

specs0171739 AK-968/40707401 3.14 395. 32

specs4086774 AK-968/11367247 2.26 369. 33

specs4087434 AK-968/11566598 1.72 331. 34

timtt017652 ST017652 3.57 460. 35

timtt017715 ST017715 3.04 365. 36

timtt017987 ST017987 4.26 415. 37

timtt025827 ST025827 1.05 337. 38

timtt4016474 ST4016474 0.39 334.

TABLE 13 ligase 180 specs0112290 85% COMP NAME IDNUMBER MW logP c . . .  1

specs0112290 AG-205/15425118 301. 2.44 1.0  2

chemdiv0310191 8011-6630 362. 4.41 25.  3

chemdiv0310192 8011-6631 362. 4.45 26.  4

chemdiv0310201 8011-6652 300. 2.80 85.  5

chemdiv0310393 8011-7163 286. 2.46 12.  6

chemdiv0310717 8011-7952 270. 2.80 9.0  7

chemdiv0310830 8011-8313 348. 3.82 79.  8

chemdiv0310998 8011-9000 362. 4.25 10.  9

chemdiv0311207 8011-9694 348. 4.11 11. 10

chemdiv0311221 8011-9716 378. 4.07 12. 11

chemdiv0311222 8011-9717 427. 4.91 80. 12

chemdiv0311440 8012-0207 405. 3.40 13. 13

chemdiv0311639 8012-0573 378. 4.11 12. 14

chemdiv0312704 8012-3473 405. 3.44 13. 15

chemdiv0313234 8012-4572 378. 3.82 81. 16

chemdiv0313235 8012-4573 427. 4.91 82. 17

chemdiv0314847 8012-7734 362. 4.45 35. 18

chemdiv0314848 8012-7735 315. 2.77 36. 19

chemdiv0315436 8012-8805 335. 3.07 37. 20

chemdiv0315593 8012-9041 392. 4.41 38. 21

chemdiv0315842 8012-9471 382. 4.71 90. 22

chemdiv0315970 8012-9712 376. 4.74 39. 23

chemdiv166863 8004-3116 359. 4.10 3.0 24

chemdiv167327 8004-6065 391. 4.12 91. 25

chemdiv4081327 8013-0295 382. 4.70 92. 26

chemdiv4081378 8013-0379 382. 4.74 90. 27

chemdiv4081817 8013-1129 382. 4.74 90. 28

chemdiv4082010 8013-1437 376. 4.75 42. 29

chemdiv4082586 8013-2547 270. 2.67 95. 30

chemdiv4082587 8013-2548 362. 4.31 96. 31

chemdiv4082589 8013-2550 364. 4.75 43. 32

chemdiv4082929 8013-3105 378. 4.88 44. 33

chemdiv4085402 8014-2098 256. 2.47 2.0 34

maybridge0403444 BTB = 04948 242. 1.81 76. 35

maybridge0403698 BTB = 05308 361. 2.76 5.0 36

specs0113723 AG-205/33687036 393. 4.73 77. 37

specs0137866 AG-690/36533026 419. 3.92 21. 38

specs4047979 AG-690/11629040 270. 2.77 24. 39

timtt049828 ST049828 362. 4.41 60. 40

timtt049829 ST049829 362. 4.45 61. 41

timtt050161 ST050161 362. 4.45 68.

TABLE 14 mol COMP_NAME IDNUMBER MW logP  1

specs4002175 AC-907/34129012 246.29 1.84  2

chembridge0120016 6636907 310.38 4.05  3

chembridge0245406 5808234 233.29 2.14  4

chembridge0273454 6955471 275.38 3.11  5

chembridge0277001 6988183 297.38 3.66  6

chembridge0401734 7934912 258.3 1.8  7

chembridge096171 5940801 302.18 3.36  8

chemdiv0281324 4065-0116 233.29 2.14  9

chemdiv178113 8088-5795 281.77 3.37 10

maybridge0415961 GK = 03672 302.18 3.32 11

maybridge0438482 RDR = 01717 219.27 2.17 12

specs0142965 AG-690/40701022 247.32 2.44 13

specs4001624 AB-323/13887140 230.23 1.05 14

specs4002174 AC-907/34129010 214.23 0.988 15

specs4002176 AC-907/34129013 230.23 0.68 16

specs4018988 AG-205/07664054 245.24 0.375 17

specs4037037 AG-205/41280558 297.38 3.66 18

specs4064355 AG-690/40700626 258.3 1.8

TABLE 15 ligase_197_specs4028351_similar80 COMP_NAME IDNUMBER MW logP clus  1

197_specs4028351 AG-205/32243059 308 1.87 1  2

amb_a0005908 5908 308 1.87 17  3

amb_a0034227 34248 368 2.42 23  4

amb_a0041542 6653 308 1.87 26  5

amb_b0092949 844 321 2.18 2  6

amb_c0086983 BAS = 1172053 368 2.42 29  7

amb_c0104748 A2485/0105630 307 1.85 3  8

amb_c0112456 3380 321 2.18 2  9

amb_d0002779 BAS = 1172053 368 2.42 29 10

amb_d030490 A2485/0105630 307 1.85 3 11

amb_e0047907 6653 308 1.87 26 12

chembridge0153135 5928213 308 1.87 26 13

chembridge0153147 5928363 294 1.66 32 14

chembridge0165304 6057299 370 3.53 33 15

chembridge0165868 6062534 368 2.42 23 16

chembridge0211898 6534037 280 1.44 34 17

chembridge0255068 6243354 307 1.85 3 18

chembridge0256700 6515124 308 1.87 1 19

chembridge0257432 6558640 308 1.87 17 20

chemdiv030171 8002-7527 368 2.42 23 21

chemdiv033499 8003-7465 308 1.87 1 22

chemdiv033518 8003-7503 308 1.87 17 23

chemdiv037027 8004-7581 370 3.53 35 24

chemdiv042175 8005-9535 308 1.87 17 25

chemdiv4080846 8011-5630 307 1.85 3 26

specs4027891 AG-205/14552050 307 1.85 3 27

timtt045056 ST045056 307 1.85 3 28

timtt057325 ST057325 308 1.87 1 29

timtt057327 ST057327 308 1.87 17 30

timtt204715 ST204715 308 1.87 17 31

timtt207481 ST207481 368 2.42 29

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All of the cited references are hereby specifically incorporated by reference in their entirety.

While the invention has been described with reference to certain particular embodiments thereof, the invention is not to be limited to the specific embodiments described and those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention.

TABLE 3 192 Compounds Tested List of 192 compounds that have been assayed 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 32 33 34 35 36 37 38 39 40 41 42 43 45 46 47 48 49 51 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 75 76 77 78 79 80 81 82 83 84 85 86 88 89 90 92 93 94 95 96 97 99 100 101 102 103 104 105 106 107 108 109 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 132 135 136 137 138 139 140 141 142 143 150 151 153 159 161 162 165 16 170 172 173 175 176 177 178 179 180 181 182 183 184 185 186 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 234

TABLE 4 Molecular properties of the 15 active compounds, their experimental inhibition rate targeting Ligl and the growth inhibition. hLigI_Inh % Growth_Inh % (50 μM) Cmpd Rot Ring MW Ha Hd LogP (100 μM) MCF10A HCT116 25 1 2 190.13 2 1 0.24 59.3 ± 7.5  32 6 3 385.38 6 1 2.10 83 4 ± 3.1  64 4 2 288.65 2 0 2.15 60.6 ± 0.54 40 67 6 2 485.11 2 2 4.91 78.3 ± 8.5  70 70 82 4 2 308.66 3 2 4.84 59.8 ± 1.7  70 40 113 8 2 475.38 5 2 1.42 53.4 ± 7.6  123 4 1 228.32 4 0 1.51 66.9 ± 3.5   175* 1 2 173.15 2 0 1.12 54.2 * 16.5  180* 2 3 301.21 2 1 2.44 58.3 * 6.0  70 70 189 2 2 244.28 3 i 1.96 68.8 ± 4.9  70 70 190 2 3 268.23 5 0 1.78 52.5 * 6.6  70 40  192* 2 3 274.28 2 1 1.56 68.2 * 1.5  70 197 6 2 308.31 0 0 1.87 91.0 * 6.9  200 2 2 212.2  1 0 3.04 50.7 * 10.9  202* 4 4 390.42 1 0 5.08 95.0 * 6.0  50 *= compounds are those that also inhibit T4 DNA ligase by >50% Rot is the number of rotatable bonds, Ring is the number of rings, MW is the molecular weight; Ha is the number of H-bond acceptors, Hd is the number of H-bond donors, LogP is the octanol/water partition coefficient. DNA joining and cell culture assays were performed as described. Inhibition of joining and proliferation is expressed as a percentage of values obtained with DMSO alone.

TABLE 5 % activity Inhibition % growth inhibition hLigI hLigIII hLigIV LigT4 MCF10A HCT116 MCF10A HCT116 Drug # 100 uM gel 100 uM gel 100 uM gel 100 uM 50 uM [2 Gy] 5-15 uM A# Drug that inhibit human DNA Ligases but not T4 DNA ligase or more than T4 A1# Common inhibitors for hLig1, hLigIII, and hLigIV 64 60.6 ± 0.54 50 48.5 ± 0.1  +++ 51.9 ± 1.2  66 18.4 ± 8.4  40 70 189 68.8 ± 4.9  75 52.6 ± 4.8  ++ >50 80 3.2 ± 1.9 70 70 197 91.0 ± 6.9  98 96.5 ± 3.5  +++ >60 100 24.4 ± 5.8  A2# Common inhibitors for hLig1 and hLigIII 67 78.3 ± 8.5  73 88.2 ± 11.6 ++ Ø   0 ± 10.5 70 70 ✓ 200 50.7 ± 10.9 53 59.5 ± 5.2  ++ Ø 0 70 70 ✓ 82 59.8 ± 1.7  76 29.24 ± 7.30  + Ø 13.9 ± 11.4 70 40 25 59.3 ± 7.5  +++ 72.8 ± 4.8  ++ Ø 12.9 ± 6.3  213 36.4 ± 8.6  + 25.2 ± 0.46 ± Ø 70 70 207 20.7 ± 19.0 + 25.8 ± 10.7 ± Ø 10.0 ± 2.0  A3# Common inhibitors for hLig1 and hLigIV 113 53.4 ± 7.6  +++ Ø Ø >50 83 5.4 ± 4.5 A4# Inhibitors unique to hLig1 184 37.7 ± 12.4 ND Ø Ø Ø 0.42 ± 1.4  70 40 ✓ 190 52.5 ± 6.6  +++ Ø Ø Ø  5.4 ± 0.74 40 40 1 40.3 ± 8.6  +++ Ø Ø Ø   0 ± 3.8 43 29.2 ± 3.5  + Ø Ø Ø 0 70 70 151 25.9 ± 1.2  + Ø Ø Ø 70 70 ✓ ✓ 35 38.7 + Ø ND Ø 3.7 ± 2.3 37 25.2 ± 14.1 + Ø ND Ø 3.7 ± 0   47 24.2 ± 4.3  + Ø ND Ø 2.2 ± 0.7 51 24.3 ± 13.5 + Ø ND Ø   0 ± 2.6 53 36.4 ± 4.7  + Ø ND Ø 3.7 ± 4.8 54 32.2 ± 10.5 + Ø ND Ø  6.9 ± 0.45 55 7.5 ± 9.3 + Ø ND Ø 0.58 ± 3.1  90 20.7 ± 4.8  + Ø ND Ø 1.6 ± 3.1 103 23.1 ± 1.8  + Ø ND Ø 6.2 ± 4.3 193 28.2 ± 34.4 + Ø ND Ø 0.44 ± 2.1  A5# inhibitors unique to hLigIII 209 Ø ND 70.5 ± 2.6  ++ Ø 46 Ø ND 21.57 ± 8.56  + Ø A6# inhibitors unique to hLigIV 215 Ø ND Ø ND 60.7 ± 1.3  53 Ø 93 Ø ND Ø ND 37.6 ± 6.1  Ø Ø 122 Ø ND Ø ND 16.1 ± 19.1 50 Ø

TABLE 5A In Vitro and In Vivo properties of identified human DNA ligase inhibitors. In vitro In vivo % inhib Cell growth inhb hLigI hLigIII hLigIV T4 Extract MCF10A HCT116 cell survival Cmpd 100 (μM) Mechanism 100 (μM) % Inhb at 50 (μM) EC50 (μM) Inhibit I, III, IV 64 61 ± 1 49 ± 1 52 ± 1 18 ± 8  1st step 40 IR sensitive, EC50~50 189 69 ± 5 53 ± 5 41 ± 6 3 ± 2 2nd, 3rd step BER/NHEJ 70 70 IR sensitive, EC50~50 197 91 ± 7 96 ± 4 57 ± 3 24 ± 6  3rd step no activity Inhibit I, III 67 78 ± 8  88 ± 12 0  0 ± 11 3rd step BER 70 70 IR, MMS sensitive, EC50~10 200  51 ± 11 60 ± 5 0 0 metabolite active 25 59 ± 8 73 ± 5 0 13 ± 6  213 36 ± 9 25 ± 1 0 0 70 70 Inhibit I, IV 113 53 ± 8 0 83 ± 5 5 ± 5 I specific 82 60 ± 2 29 ± 7 0 14 ± 11 3rd step BER 70 40 cytostatic, G1, EC50~50 151 26 ± 1 0 0 0 BER 70 70 MMS sensitive, EC50~10 184  38 ± 12 0 0 1 ± 1 70 40 EC50~10 190 53 ± 7 0 0 5 ± 1 40 40 1 40 ± 9 0 0 0 ± 4 43 29 ± 4 0 0 0 70 70 III specific 209 0 71 ± 3 0 0 EC50 > 100 IV specific 215 0 0 61 ± 1 0 EC50 > 100 122 0 0 50 ± 5 0 EC50 > 100 93 0 0 37 ± 6 0

TABLE 6 Compounds that decreased cell growth at low concentrations: % of inhibition @ IC₅₀ (μM) 100 μM (Helen's data) Compound MCF10A HCT116 hLigI hLigIII hLigIV 16^(t) 6 NI NI NI NI 67 4 8 78 88 NI 78 7.5 10 ND >20 <20 151 6 22 25 NI NI 165 3 NI NI NI 37.2 180 3 12 58 >40 NI 195* 3 NI 26 24 NI 200 ID (5) ID (10) 51 60 NI Radiosensitizers: % of inhibition @ IC₅₀ ratios 100 μM (Helen's data) Compound MCF10A HCT116 hLigI hLigIII hLigIV 64 1.2 NI 61 49 52 151 1 2 26 NI NI 105^(t) 1 1.3 NI NI NI For 159 no IC₅₀ was achieved, but only slight sensitization to IR was observed. 159 did not inhibit human ligases in vitro. Compounds that sensitized cells to MMS: % of inhibition @ Ratios of IC₅₀ 100 μM (Helen's data) Compound MCF10A HCT116 hLigI hLigIII hLigIV 16^(t) 1 NI NI NI NI 67 1.2 1.4  78 88 NI 78 1.5 1  ND >20 <20 151 1.8 1.7  25 NI NI 159^(t) ND 2** NI NI NI 165 1 NI NI NI 37.2 180 1 ND 58 >40 NI 195* 1.2 NI 26 24 NI 198^(t) NI 2** NI NI NI 200 ID (1) ID (1) 51 60 NI 214* NI ID (2) ND 42 40 IC₅₀ ratio: IC₅₀ value derived from growth curve of cells treated with compound alone compared to the IC₅₀ derived from growth curve of cells treated in combination with MMS (100 μM). Growth curves were normalized to either untreated control or control treated with MMS alone. The ratio of IC₅₀ of compounds that do not sensitize cells to MMS is equal to 1. Compounds that sensitize cells to the PARP inhibitor 3-amino benzamide (3-AB): IC₅₀ ratios of non- and % of inhibition @ 3-AB treated cells 100 μM (Helen's data) Compound MCF10A HCT116 hLigI hLigIII hLigIV 64 2  1.6** 61 49 52 67 1.7  1    78 88 NI 78 1  ND ND >20 <20 198^(t) 2** 1    NI NI NI 200 ID (1.2) ID (1) 51 60 NI 214* ID (2) ID (1.3) ND 42 40 IC50 ratio: ratio of IC₅₀ value derived from growth curve of cells treated with compound alone and IC₅₀ derived from growth curve of cells treated with compounds in combination with 3-AB (2 mM). Growth curves were normalized to either DMSO control or to DMSO and 3-AB. The ratio of IC₅₀ of compounds that do not sensitize cells to 3-AB is equal to 1. Compounds that inhibit cell growth at high concentrations (~50 μM) % of inhibition @ IC₅₀ 100 μM (Helen's data) Compound MCF10A HCT116 hLigI hLigIII hLigIV 64 50 NI 61 49 52 105^(t) NI 50 NI NI NI 159^(t) NI 50 NI NI NI 177 ND 45 48 46 NI 198^(t) NI NI NI NI NI 208 40 ND 21 24 NI 212^(t) ND NI NI NI NI 214* ID ID ND 42 40 Compounds tested in the MTT assay that at 50 μM did not or only slightly inhibit cell proliferation of either MCF10A or HCT116: % of inhibition @ Cell line 100 μM (Helen's data) MCF10A HCT116 hLigI hLigIII hLigIV — 16^(t) NI NI NI — 64 61 49 52 79^(t) ND NI NI NI 124^(t) 124 NI NI NI 130^(t) 130 NI NI NI — 165 NI NI 37 ND 184 (30%) 38 NI NI 193^(t) ND NI NI NI — 195 26 24 NI 197 197 91 97 57 198**^(t) 198**^(t) NI NI NI 199^(t) (30%) ND NI NI NI ND 212^(t) NI NI NI *214** — NI 42 40 221^(t) ND >20 NI NI Compounds that inhibit growth of both cell lines: MCF10A and HCT116 % of inhibition @ IC₅₀ (μM) 100 μM (Helen's data) Compound MCF10A HCT116 hLigI hLigIII hLigIV 67 4 8 78 88 NI 78 7.5 10 ND >20 <20 151 6 22 25 NI NI 180 3 12 58 >40 NI 200 ID (5) ID (10) 51 60 NI Compounds active on MCF10A cell line only: IC₅₀ (μM) % of inhibition @ 100 μM (Helen's data) Compound MCF10A hLigI hLigIII hLigIV 16^(t) 6 NI NI NI 64 50 61 49 52 165 3 NI NI 37 195 3 26 24 NI 208 40 21 24 NI Compounds active on HCT116 cells more than on MCF10A: % of inhibition @ IC₅₀ (μM) 100 μM (Helen's data) Compound HCT116 hLigI hLigIII hLigIV 105^(t) ~50 NI NI NI 159^(t)  50 NI NI NI 198^(t) ** NI NI NI 214* ID NI 42 40 (10-50) Compounds active in vitro that decreased cell growth of either MCF10A or HCT116 cell line: % of inhibition @ IC₅₀ 100 μM (Helen's data) Compound MCF10A HCT116 hLigI hLigIII hLigIV 64 50 NI 61 49 52 67 4 8 78 88 NI 78 7.5 10 ND >20 <20 151 6 22 25 NI NI 177 ND 45 48 46 NI 165 3 NI NI NI 37.2 180 3 3 58 >40 NI 195* 3 NI 26 24 NI 200 ID ID 51 60 NI (10-150) (10-150) 208 40 ND 21 24 NI 214* NI ID ND 42 40 (10-50) Sensitizers to MMS and 3-AB MMS 3-AB % of inhibition @ IC₅₀ ratios IC₅₀ ratios 100 μM (Helen's data) Compound MCF10A HCT116 MCF10A HCT116 hLigI hLigIII hLigIV 64 — 2** 2  1.6 61 49 52 67^(tt) 1.2 1.4  1.7  1 78 88 NI 78^(tt) 1.5 1  1  ND ND >20 <20 151^(tt) 1.8 1.7  ND ND 25 NI NI 159^(t) Ni 2*  ND ND NI NIt NI 195* 1.2 NI (1) ND ND 26 24 NI 198^(t) NI (1)t 2** 2** 1 NI NIt NI 214* NI (1) ID (2) 2  1 ND 42 40 Total # of compounds tested on either cell line: 24 out of 192 t indicates compounds that are inhibit cell growth but did not show any activity to inhibit ligases under cell-free conditions. tt indicates are the most active agents as tested on MCF10A and HCT116 cell lines *compounds that are insoluble, form crystals in media **compounds that alone at 50 μM did not inhibit cell growth by 50%, however sensitized cells to MMS or 3-AB #198** active at high concentrations (50 μM) sensitized MCF10A cells to 3-AB, and HCT116 cells to MMS; #214** sensitized MCF10A cells to 3-AB, and HCT116 to MMS. 214 is highly insoluble and undergoes self degradation in DMSO (MS data, BD). ND- no data; ID- inconsistent data; NI- no inhibition

TABLE 7 Similarity between 10 active compounds (bold numbers) based on the Tanimoto Index (Tc %) 25 64 67 82 113 123 189 190 197 200 25 100 64 39 100 67 38 53 100 82 42 53 69 100 113 22 34 45 30 100 123 15 32 16 16 22 100 189 40 40 37 43 24 30 100 190 43 28 32 39 18 19 52 100 197 40 35 24 21 18 20 18 22 100 200 40 19 25 27 13 4 25 35 35 100 

We claim:
 1. A method of ameliorating neuroblastoma exhibiting an altered DNA damage response in a patient in need comprising administering to said patient an effective amount of an anti-cancer compound according to the chemical structure as set forth below:

in combination with a second cancer treatment comprising a DNA damaging agent.
 2. The method according to claim 1 wherein said DNA damaging agent is ionizing radiation, a PARP inhibitor or a DNA alkylating agent.
 3. The method according to claim 2 wherein said DNA damaging agent is a PARP inhibitor.
 4. The method according to claim 3 wherein said PARP inhibitor is 3-aminobenzamide.
 5. The method of claim 1, wherein said DNA damaging agent is administered at the same time as administering said compound.
 6. The method of claim 1, wherein said DNA damaging agent is administered prior to administering said at least one compound.
 7. The method of claim 1, wherein said DNA damaging agent is administered after administering said at least one compound. 